Compositions and methods for treatment of cancer

ABSTRACT

The present invention concerns compositions and methods for the treatment of disorders characterized by the over-expression of an LIV-1. More specifically, the compositions include DNA and amino acid sequences of an LIV-1, antibodies to an LIV-1, and methods for the treatment of a mammal susceptible to or diagnosed with cancer wherein an LIV-1 is overexpressed.

FIELD OF THE INVENTION

[0001] The present invention concerns compositions and methods for thetreatment of disorders characterized by the overexpression of a LIV-1gene product in tumors. The compositions comprise a nucleic acid, apolypeptide encoded by the nucleic acid, and a compound, preferably anantibody or fragment thereof, that binds to the polypeptide, preferablybinding to the extracellular domain of LIV-1 polypeptide.

BACKGROUND OF THE INVENTION

[0002] Breast cancer is a common and devastating form of cancer,affecting millions of women per year throughout the world. Many breasttumors are estrogen sensitive and frequently treatable with compoundsthat interfere with estrogen binding to estrogen receptors (ERs)expressed on the breast tumor tissue. Detecting the level of ERexpression and sensitivity to estrogen stimulation is useful fordetermining that antihormone-type chemotherapy may succeed in aparticular patient.

[0003] The overexpression of estrogen-inducible genes, pLIV-1 and pLIV2(also designated pS2), occurs in some breast tumors which also expressthe estrogen receptor, (Manning D. L. et al. European J. Cancer 29A(10):1462-1468 (1993): Manning. D. L. et al., European J. Cancer30A(5):675-678 (1994); Manning. D. L. et al. Acta Oncologica 34(5):641-646 (1995); Manning, D. L. et al., U.S. Pat. No. 5,693,465).Expression of pLIV-1, but not pS2, is associated with metastasis ofbreast cancer cells to regional lymph nodes (Manning et al. U.S. Pat.No. 5,692,465).

[0004] In addition, the pathogenesis of various human malignancies,including breast cancer, is affected by proto-oncogenes that encodegrowth factors and growth factor receptors. Human ErbB2 gene (erbB2,also known as her2, or c-erbB-2), which encodes a 185-kd transmembraneglycoprotein receptor (ErbB2, also known as HER2 or p185^(HER2)) relatedto the epidermal growth factor receptor (EGFR), is overexpressed inabout 25% to 30% of human breast cancer (Slamon et al., Science235:177-182 [1987]: Slamon et al. Science 244:707-712 [1989]).

[0005] Several lines of evidence support a direct role for ErbB2 in thepathogenesis and clinical aggressiveness of ErbB2-overexpressing tumors.The introduction of ErbB2 into non-neoplastic cells causes theirmalignant transformation (Hudziak et al. Proc. Natl. Acad. Sci. USA84:7159-7163 [1987]; DiFiore et al., Science 237:78-182 [1987]).Transgenic mice that express ErbB2 develop mammary tumors (Guy et al.,Proc. Natl. Acad. Sci. USA 89:10578-10582 [1992]). ErbB2 overexpressionis commonly regarded as a predictor of a poor prognosis in humans,especially in patients with primary disease that involves axillary lymphnodes (Slamon et al. [1987] and [1989], supra: Ravdin and Chamness, Gene159:19-27 [1995]; and Hynes and Stern, Biochim Biophys Acta 1198:165-184[1994]).

[0006] Antibodies directed against human erbB2 protein products(anti-ErbB2 antibodies) and against proteins encoded by the ratequivalent of the erbB2 gene (neu) (anti-neu protein antibodies)down-modulate cell surface expression of p185 on B104-1-1 cells (NIH-3T3cells transfected with the neu proto-oncogene) and inhibit colonyformation of these cells, Drebin et al., Cell 41:695-706 (1985).Biological effects of anti-neu protein antibodies are reviewed in Myerset al., Meth. Enzym. 198:277-290 (1991). See also WO94/22478 publishedOct. 13, 1994.

[0007] The anti-ErbB2 antibody, 4D5, exhibited anti proliferativeeffects on the SKDR3 human breast tumor cell line, inhibiting cellularproliferation by approximately 56%, and sensitizingp185^(erbB2′)-overexpressing breast tumor cell lines to the cytotoxiceffects of TNF-α. See Hudziak et al., Mol. Cell. Biol.9(3):1165-1172(1989). See also WO89/06692 published Jul. 27, 1989. Theanti-ErbB2 antibodies discussed in Hudziak et al. are furthercharacterized in Fendly et al. Cancer Research 50:1550-1558 (1990);Kotts et al. In Vitro 26(3):59A (1990): Sarup et al. Growth Regulation1:72-82 (1991): Shepard et al. J. Clin. Immunol. 11(3): 117-127 (1991):Kumar et al. Mol. Cell. Biol. 11(2):979-986 (1991); Lewis et al. CancerImmunol. Immunother. 37:255-263 (1993); Pietras et al. Oncogene9:1829-1838 (1994); Vitetta et al. Cancer Research 54:5301-5309 (1994);Sliwkowski et al. J. Biol. Chem. 269(20):14661-14665 (1994): Scott etal. J. Biol. Chem. 266:14300-5 (1991); and D'souza et al. Proc. Natl.Acad. Sci. 91:7202-7206 (1994).

[0008] ErbB2 overexpression is also linked to sensitivity and/orresistance to hormone therapy and chemotherapeutic regimens, includingCMF (cyclophosphamide, methotrexate, and fluoruracil) and anthracyclines(Baselga et al., Oncology 11(3 Suppl 1):43-48 [1997]). Despite theassociation of ErbB2 overexpression with poor prognosis, the odds ofHER2-positive patients responding clinically to treatment with taxaneswere greater than three times those of HER2-negative patients (Ibid),rhuMab HER2 was shown to enhance the activity of paclitaxel (TAXOL®) anddoxorubicin against breast cancer xenografts in nude mice injected withBT-474 human breast adenocarcinoma cells, which express high levels ofHER2 (Baselga et al. Breast Cancer, Proceedings of ASCO, Vol. 13.Abstract 53 [1994]).

[0009] Because breast and other cancers pose constant threats to health,there is a continuing need to develop treatments for cancers by usingmethods that target cancer cells without simultaneously harming largenumbers of non-cancerous cells, thereby limiting adverse side effectsassociated with traditional cancer chemotherapy.

SUMMARY OF THE INVENTION

[0010] The present invention relates to the discovery of a uniqueprotein. LIV-1-164647, that is overexpressed in some tumor tissues, suchas in prostate, colon, lung, breast, and a population of breast tumorsthat overexpress LIV-1-164647, but do not overexpress ErbB2. The presentinvention further relates to nucleic acid sequences and amino acidsequences having homology to herein disclosed LIV-1 gene sequence(designated DNA 164647) and the amino acid sequence of LIV-1 proteinencoded by DNA 164647. Applicants' discovery that LIV-1-164647 isoverexpressed in tumor cells led to the additional discoveries ofcompositions for detection and treatment of tumor cells and methods ofcarrying out such detection and treatment.

[0011] In one aspect, the present invention relates to a nucleic acidsequence having homology to the nucleic acid sequence of DNA 164647 (SEQID NO:3 (coding strand)), or a portion thereof. Preferably the homologyis at least approximately 80% homology, more preferably at leastapproximately 90%, still more preferably at least approximately 95%, andmost preferably at least approximately 97% homology. Preferably, thenucleic acid of the invention encodes an aqueous soluble extracellulardomain (ECD) that is at least 80% homologous to the DNA 164647 (SEQ IDNO:3) from approximately nucleic acid 73 to approximately 1060.Preferably, the homologous nucleic acid of the invention hybridizesunder stringent conditions to a 30 nucleic acid or longer portion of thenucleic acid sequence of DNA 164647 (SEQ ID NO:3) or its complementarysequence, preferably hybridizing under stringent conditions to a 30nucleotide regions from nucleotide 440 to and including nucleotide 470of SEQ ID NO:3, or its complementary sequence. In a related embodiment,the homologous nucleic acid of the invention comprises a nucleic acidsequence comprising a sequence that is at least 50%, preferably at least80%, more preferably at least 90% homologous to the sequence fromnucleotide 446 to and including nucleotide 463 of SEQ ID NO:3, or asequence from nucleotide 2297 to and including 2337 of SEQ ID NO:3, orboth sequences. Most preferably, the isolated nucleic acid comprises asequence from nucleotide 446 to and including nucleotide 464 and/or fromnucleotide 2297 to and including 2337. According to the presentlydisclosed invention, the isolated nucleic acid of the inventioncomprises a sequence having at least 65%, preferably at least 75%, morepreferably at least 85%, still more preferably at least 90%, and mostpreferably at least 96% homologous to a sequence from approximatelynucleotide 412 to and including nucleotide 477 of SEQ ID NO:3, or itscomplementary sequence. Preferably, the sequence encodes ahistidine-rich region of an antigenic polypeptide, preferably an ECD.

[0012] In another aspect, the present invention relates to an isolatedpolypeptide comprising an amino acid sequence having homology to theamino acid sequence (SEQ ID NO:4), or a fragment thereof, encoded by orwithin DNA 164647, designated herein as LIV-1-164647. Preferably thehomology is at least approximately 80% homology, more preferably atleast approximately 90%, still more preferably at least approximately95%, and most preferably at least approximately 97% homology.Preferably, a LIV-1-164647 amino acid sequence of the invention is anaqueous soluble ECD homologous to amino acid 1 to approximately aminoacid 327 or a fragment thereof comprising at least 10 amino acids. Theregion of the ECD is readily determined from a standard hydropathy plotindicating the relatively more hydrophilic region N-terminal of ahydrophobic transmembrane region. In a related embodiment, thehomologous amino acid sequence of the invention comprises a sequencefrom amino acid 126 to and including amino acid 132 of SEQ ID NO:4(specifically, the amino acid sequence HDHHSHH (SEQ ID NO:17)), or asequence from amino acid 743 to and including amino acid 755 of SEQ IDNO:4 (specifically, the amino acid sequence SIFEHKIVFRINF (SEQ IDNO:18), or both sequences. The present invention further includes anisolated polypeptide comprising an amino acid sequence having at least50%, preferably at least 80%, more preferably at least 90% homologous toSEQ ID NO:17. The present invention still further includes an isolatedpolypeptide comprising an amino acid sequence having at least 20%, morepreferably at least 50%, still more preferably at least 80%, and mostpreferably at least 90% homology to SEQ ID NO:18. In still anotherembodiment, the invention includes an isolated nucleic acid of SEQ IDNO:3 and an isolated polypeptide of SEQ ID NO:4. The present inventionfurther includes an isolated polypeptide comprising an amino acidsequence having at least 65%, preferably at least 75%, more preferablyat least 85%, still more preferably at least 90%, and most preferably atleast 96% to a sequence from amino acid 114 to and including amino acid135 of SEQ ID NO:4. The amino acid sequence from amino acid 114 to 135is designated SEQ ID NO:19. A still further embodiment includes anisolated polypeptide comprising SEQ ID NO:17 and/or SEQ ID NO:18.

[0013] In still another embodiment, the invention includes an isolatedpolypeptide comprising an amino acid sequence wherein the sequence is atleast 98% homologous to the sequence from amino acid 1 to and includingamino acid 327 of SEQ ID NO:4, more preferably comprising the ECD ofLIV-1-164647. Most preferably, the sequence comprises SEQ ID NO:17,forms a portion of an extracellular domain (ECD), preferably the ECD ofLIV-1-164647. The term “portion” as used herein refers to a sequencethat comprises at least 7 amino acids of the ECD of LIV-1-164647 fromamino acid 1 to and including amino acid 327 of SEQ ID NO:4.

[0014] In another embodiment, the present invention concerns an antibodywhich specifically binds a LIV-1 polypeptide. Preferably, the antibodyis a monoclonal antibody. More preferably, the antibody is a humanantibody or a humanized antibody. In one embodiment, the antibodyreduces activity of overexpressed LIV-1 polypeptide in a cell. Inanother aspect, the antibody is a monoclonal antibody, which preferablyhas nonhuman complementarity determining region (CDR) residues and humanframework region (FR) residues. The antibody may be labeled and may beimmobilized on a solid support. In a further aspect, the antibody is anantibody fragment, a single-chain antibody, or an anti-idiotypicantibody. Preferably, a LIV-1-binding antibody of the invention bindsspecifically to a polypeptide having at least approximately 80%homology, more preferably at least approximately 90% homology, stillmore preferably at least approximately 95% homology, and most preferablyat least approximately 97% homology to the LIV-1 ECD nucleic acidsequence, or a fragment thereof, encoded within the ECD coding region(nucleotides 1-1000) of DNA 164647 (SEQ ID NO:3). More preferably, aLIV-1-binding antibody of the invention binds specifically to apolypeptide having at least 80% homology, more preferably at leastapproximately 90% homology, still more preferably at least approximately95% homology, and most preferably at least approximately 97% homology tothe amino acid sequence of LIV-1 ECD (amino acids 1-327 of SEQ ID NO:4).In a preferred embodiment, the present invention concerns an isolatedantibody which specifically binds a LIV-1 polypeptide encoded by anucleic acid sequence comprising a nucleic acid sequence having at least65%, preferably at least 75%, more preferably at least 85%, still morepreferably at least 90%, and most preferably at least 96%, homology to asequence from nucleotide 446 to and including nucleotide 463 of SEQ IDNO:3, or the nucleic acid sequence from 2297 to and including 2337 ofSEQ ID NO:3, or both nucleic acid sequences. In another preferredembodiment, the present invention concerns an isolated antibody whichspecifically binds a LIV-1 polypeptide comprising the amino acidsequence having at least 65%, preferably at least 75%, more preferablyat least 85%, still more preferably at least 90%, and most preferably atleast 96% homology to a sequence from amino acid 126 to and includingamino acid 132 of SEQ ID NO:4, or the amino acid sequence from aminoacid 743 to and including amino acid 755 of SEQ ID NO:4, or both aminoacid sequences.

[0015] In still another embodiment, the invention concerns an antibody,preferably a monoclonal antibody, that specifically binds the sameepitope of LIV-1, preferably LIV-1-1164647, that is bound by any one ofthe monoclonal antibodies produced by the hybridoma cell lines depositedwith the American Type Culture Collection (ATCC) as disclosed herein.

[0016] In a further embodiment, the invention includes an antibody thatbinds to a polypeptide comprising a sequence from amino acid 1 to andincluding amino acid 147 of SEQ ID NO:4. In another embodiment, theantibody binds a polypeptide comprising amino acid 148 to and includingamino acid 298 of SEQ ID NO:4. Preferably, the antibodies are monoclonalantibodies. More preferably, the monoclonal antibodies are humanantibodies or humanized antibodies.

[0017] In another aspect, the invention concerns a compositioncomprising an antibody which binds LIV-1 polypeptide in an admixturewith a pharmaceutically acceptable carrier. In one aspect, thecomposition comprises a therapeutically effective amount of theantibody. In another aspect, the composition comprises a further activeingredient, which may, for example, be a further antibody or a cytotoxicor chemotherapeutic agent. Preferably, the composition is sterile.

[0018] In a further embodiment, the invention concerns a nucleic acidencoding an anti-LIV-1 antibody according to the invention, and vectorsand recombinant host cells comprising such nucleic acid.

[0019] In a still further embodiment, the invention concerns a methodfor producing an anti-LIV-1 antibody by culturing a host celltransformed with nucleic acid encoding the antibody under conditionssuch that the antibody is expressed, and recovering the antibody fromthe cell culture.

[0020] The invention further concerns antagonists and agonists of aLIV-1 polypeptide, which antagonists inhibit one or more of thefunctions or activities of the LIV-1 polypeptide and which agonistsmimic one or more of the functions or activities of the LIV-1polypeptide. Preferably the LIV-1 polypeptide is the LIV-1-164647polypeptide whose functions or activities are antagonized or agonized.

[0021] In another embodiment, the invention concerns a method fordetermining the presence of a LIV-1 polypeptide or fragment thereofcomprising exposing a cell suspected of containing the LIV-1 polypeptideto an anti-LIV-1 antibody of the invention and determining binding ofthe antibody to the cell.

[0022] In yet another embodiment, the present invention concerns amethod of diagnosing tumor in a mammal, comprising detecting the levelof expression of a gene encoding a LIV-1 polypeptide in a test sample oftissue cells obtained from the mammal, and in a control sample of knownnormal tissue cells of the same cell type, wherein a higher expressionlevel in the test sample indicates the presence of tumor in the mammalfrom which the test tissue cells were obtained.

[0023] In another embodiment, the present invention concerns a method ofdiagnosing tumor in a mammal, comprising contacting an anti-LIV-1antibody with a test sample of tissue cells obtained from the mammal,and detecting the formation of a complex between the anti-LIV-1 antibodyand the LIV-1 polypeptide in the test sample. The detection may bequalitative or quantitative, and may be performed in comparison withmonitoring the complex formation in a control sample of known normaltissue cells of the same cell type. A larger quantity of complexesformed in the test sample indicates the presence of tumor in the mammalfrom which the test tissue cells were obtained. The antibody preferablycarries a detectable label. Complex formation can be monitored, forexample, by light microscopy, flow cytometry, fluorimetry, or othertechniques known in the art.

[0024] For the methods of diagnosing, the test sample is usuallyobtained from an individual suspected to have neoplastic cell growth orproliferation (e.g. cancerous cells).

[0025] In another embodiment, the invention involves a method ofdiagnosing breast tumor tissue as tissue that overexpresses LIV-1protein and expresses normal levels of ErbB2. The method comprisesproviding a test sample of tissue suspected of being cancerous,contacting the test sample with an antibody to the naturally occurringform of the LIV-1 gene product, contacting the same or duplicate testsample with an anti-ErbB2 antibody, detecting the relative binding ofthe antibodies to the test sample compared to a control sample, wherethe control may be a positive control, a negative control, or both. Atest sample that overexpresses LIV-1 gene product (relative to normaltissue), but does not overexpress ErbB2 protein (relative to normaltissue), is diagnosed as a member of the population of breast tumors tobe treated by the compositions and methods of the invention. Useful inthe diagnostic assay method of the invention are anti-ErbB2 antibodiesthat bind the extracellular domain of the ErbB2 receptor, and preferablybind to the epitope 4D5 or 3H4 within the ErbB2 extracellular domainsequence. Mole preferably, the antibody is the antibody 4D5. Otherpreferred ErbB2-binding antibodies include, but are not limited to,antibodies 7C2, 7F3, and 2C4. Information regarding anti-ErbB2antibodies is found, for example, in Hudziak, R. M. et al. U.S. Pat. No.5,772,997, incorporated herein by reference in its entirety.

[0026] In another aspect, the present invention concerns a cancerdiagnostic kit, comprising an anti-LIV-1-164647 antibody and a carrier(e.g. a buffer) in suitable packaging. The kit preferably containsinstructions for using the antibody to detect the LIV-1 polypeptide.

[0027] In yet another aspect, the invention concerns a method forinhibiting the growth of tumor cells comprising exposing a cell whichoverexpresses a LIV-1 polypeptide to an effective amount of an agentinhibiting the expression and/or activity of the LIV-1 polypeptide. Theagent preferably is an anti-LIV-1-164647 antibody, a small organic andinorganic molecule, peptide, phosphopeptide, antisense or ribozymemolecule, or a triple helix molecule. In a specific aspect, the agent,e.g. anti-LIV-1-164647 antibody induces cell death, or at least slowscell growth sufficiently to allow other methods of cancer treatment tobe administered. In a further aspect, the tumor cells are furtherexposed to radiation treatment and/or a cytotoxic or chemotherapeuticagent.

[0028] In yet another aspect, the invention concerns a method for thetreatment of a human patient susceptible to or diagnosed with a disordercharacterized by overexpression of LIV-1 gene product withoutoverexpression of ErbB2 receptor. In an embodiment, the method comprisesadministering a therapeutically effective amount of an anti-LIV-1polypeptide antibody, where administration may be intravenous,subcutaneous, or other pharmaceutically acceptable method of antibodydelivery. Preferably the antibody specifically binds the naturallyoccurring form of the LIV-1-164647 polypeptide, wherein the binding ispreferably to the extracellular domain or a fragment thereof.Preferably, the initial dose (or doses) as well as the subsequentmaintenance dose or doses are administered subcutaneously. Optionally,where the patient's tolerance of the anti-LIV-1 antibody is unknown, theinitial dose is administered by intravenous infusion, followed bysubcutaneous administration of the maintenance doses it the patient'stolerance for the antibody is acceptable. According to the embodiment ofthe invention, the initial dose or doses is followed by subsequent dosesof equal or smaller amounts of antibody at intervals sufficiently closeto maintain the trough serum concentration of antibody at or above anefficacious target level. Preferably, an initial dose or individualsubsequent dose does not exceed 100 mg/kg, and each subsequent dose isat least 1 μg/kg. The choice of delivery method for the initial andmaintenance doses is made according to the ability of the animal orhuman patient to tolerate introduction of the antibody into the body.Where the antibody is well-tolerated, the time of infusion may bereduced. The choice of delivery method as disclosed for this embodimentapplies to all drug delivery regimens contemplated according to theinvention.

[0029] In a further aspect, the invention provides a method of treatingLIV-1 gene product-overexpressing cancer (lacking overexpression ofErbB2) in a human patient comprising administering to the patienteffective amounts of an anti-LIV-1 antibody (which antibody preferablybinds the extracellular domain of LIV-1 gene product) and achemotherapeutic agent. In one embodiment of the invention, thechemotherapeutic agent in a toxoid including, but not limited to,paclitaxel and doxetaxel. In another embodiment, the chemotherapeuticagent is an anthracyline derivative including, but not limited to,doxorubicin and epirubicin. In still another embodiment, thechemotherapeutic agent is not administered to the patient simultaneouslywith the anti-LIV-1 antibody. One or more additional chemotherapeuticagents may also be administered to the patient.

[0030] The disorder to be treated by a method of the inventionpreferably is a benign or malignant tumor characterized by theoverexpression of the LIV-1 gene product. Preferably, the malignantcells of the tumor express approximately the same level of ErbB2 (orless) as a non-cancerous cell of the same type. For example, thedisorder to be treated is a cancer, such as breast cancer, lung cancer,and prostate cancer.

[0031] Accordingly, one aspect of the invention involves compounds thatbind to the LIV-1 protein and inhibit its activity. Preferably thecompounds bind to the extracellular region of the LIV-1 protein andinhibit its activity. In an embodiment of the invention, the inhibitingcompounds are antibodies specific to the LIV-1 gene product or fragmentsthereof. Preferably, the inhibiting compounds of the invention bindspecifically to the extracellular region of the LIV-1 protein.

[0032] In another aspect, the invention involves compounds that blockthe binding of an activating ligand of LIV-1 protein. Suchligand-blocking compounds include, but are not limited to polypeptides,proteins, antibodies and the like. Preferably the ligand-blockingcompounds specifically block the activity of a LIV-1 activating ligand.More preferably, the ligand-blocking compounds of the invention inhibitgrowth of a LIV-1-expressing cell.

[0033] In another aspect, the invention involves a method foridentifying a compound capable of inhibiting the expression and/oractivity of a LIV-1 polypeptide, comprising contacting a candidatecompound with a LIV-1 polypeptide under conditions and for a timesufficient to allow these two components to interact. In a specificaspect, either the candidate compound or the LIV-1 polypeptide isimmobilized on a solid support. In another aspect, the non-immobilizedcomponent carries a detectable label.

[0034] In yet another aspect, the invention concerns an article ofmanufacture, comprising a container; a composition within the containercomprising an anti-LIV-1 antibody that binds the LIV-1 protein(preferably binding to the extracellular domain or a fragment thereof)or binds an activating ligand of the LIV-1 protein; and optionally alabel on or associated with the container that indicates that thecomposition can be used for treating a condition characterized byoverexpression of LIV-1 without overexpression of ErbB2. According toanother embodiment of the invention, the article of manufacture furtherincludes a package insert comprising instructions to administer theanti-LIV-1 antibody subcutaneously for at least one of the doses,preferably for all of the subsequent doses following the initial dose,most preferably for all doses.

[0035] Where the methods and compositions of the invention comprise ananti-LIV-1 antibody, which specifically and preferably binds theextracellular domain of a LIV-1 gene product, or a fragment of theextracellular domain. The compositions of the invention preferablyinclude a humanized LIV-1 antibody. Thus, the invention further pertainsto a composition comprising an antibody that specifically and preferablybinds the extracellular domain of LIV-1 gene product, and pertains tothe use of the antibody for treating LIV-1+/ErbB2-expressing cancer in ahuman, e.g., LIV-1 overexpressing cancer that does not coexpress highlevels of ErbB2. Preferably the antibody is a monoclonal antibody, e.g.,humanized anti-LIV-1 monoclonal antibody that binds to the extracellulardomain (or a portion of the extracellular domain) of LIV-1 (hereinafteranti-LIV-1) The antibody may be an intact antibody (e.g., an intact IgG₁antibody) or an antibody fragment (e.g., a Fab, F(Ab)₂, diabody, and thelike). The variable light chain and variable heavy chain regions ofhumanized anti-LIV-1 antibody.

[0036] These and other advantages and features of the present inventionwill become apparent to those persons skilled in the art upon readingthe details of the invention as more fully set forth below and in theappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIGS. 1A-1C are the nucleic acid sequence (SEQ ID NO:1 (codingsequence). FIGS. 1A-1 to 1A-2) and the amino acid sequence (SEQ ID NO:2,FIG. 1B) of the LIV-1 protein. The dashed overlined portion is predictedto be a signal sequence (approximately amino acids 1 to 20). Thepredicted extracellular domain of LIV-1 protein is that portion of theLIV-1 amino acid sequence underscored by a dashed line (approximatelyamino acids 24 to 312). The predicted transmembrane domain extends fromapproximately amino acid 318 to approximately amino acid 367. Theapproximate positions of the domains were predicted using a standardhydropathy analysis program. A nucleic acid sequence (SEQ ID NO:5)encoding a portion of LIV-1 and useful in microarray analysis is shownin FIG. 1C.

[0038] FIGS. 2A-2B are the nucleic acid sequence and amino acidsequence, respectively, corresponding to DNA 164647. FIGS. 2A1-2A5 isthe nucleotide sequence of DNA 164647 that is a cDNA encoding a nativesequence LIV-1 protein. SEQ ID NO:3 is the coding strand of DNA 164647.FIG. 2B is the derived amino acid sequence (SEQ ID NO:4) of a nativeLIV-1 polypeptide encoded by DNA 164647.

[0039] FIGS. 3-1-3-11 is an alignment of SEQ ID NO:1 and SEQ ID NO:3nucleic acid sequences.

[0040] FIGS. 4-1-4-3 is an alignment of SEQ ID NO:2 and SEQ ID NO:4amino acid sequences. The sequences differ in the ECD (near amino acid130 of SEQ ID NO:4) and C-terminal region beyond about amino acid 740 ofSEQ ID NO:4. A single amino acid difference was found at amino acid 651of SEQ ID NO:4.

[0041]FIG. 5 is a flow chart illustrating the FLIP cloning method up tothe restriction digestion selection step, as described herein. Theshaded boxes flanking the vector sequence represent the target genesequences.

[0042]FIG. 6 is a graphical representation of a fluorescent activatedcell sorting (FACS) analysis in which an anti-LIV-1-164647 monoclonalantibody was shown to bind primarily to the surface of 3T3 cellstransfected with DNA 164647. The term “pRK5” refers cells transfectedwith vector lacking a LIV-1-164647 insert. The term “pRK5-LIV-1-164647”refers to cells transfected with vector expressing full lengthLIV-1-164647.

[0043]FIG. 7 is a bar graph demonstrating that the LIV-1-164647extracellular domain is expressed on the surface of cells transfectedwith DNA encoding the full-length LIV-1-164647 protein.

DESCRIPTION OF THE EMBODIMENTS

[0044] 1. Definitions

[0045] As used herein, the term “LIV-1” refers to a gene or its encodedprotein, which gene transcript is detected in above normal levels insome cancer cells. More specifically, a LIV-1 gene or protein of thepresent invention is one which is encoded by DNA 164647 (SEQ ID NO:3)and has the deduced amino acid sequence of SEQ ID NO:4. As used herein,the term LIV-1 refers to LIV-1-164647 where the disclosure refers to anucleic acid comprising at least 21 nucleotides of SEQ ID NO:3, or wherethe disclosure refers to an amino acid sequence comprising at least 7amino acids of SEQ ID NO:4 as disclosed herein. According to theinvention. LIV-1-164647 is expressed in higher than normal amounts in acell, while the gene encoding ErbB2 receptor is not expressed in higherthan normal amounts. Such higher than normal expression is termed“overexpression” of a gene or protein. The term “LIV-1” or“LIV-1-164647” may be used to refer to a LIV-1 gene or its encodedprotein. Generally, where a protein or peptide is contemplated, the term“LIV-1 protein” will be used.

[0046] The term “LIV-1 gene product” or “LIV-1 protein” refers to theexpressed protein product of the gene, preferably a polypeptide orprotein form of the gene product. According to the invention, apolypeptide or protein form of the LIV-1 gene product includes a solubleform of the gene product (i.e. the extracellular domain (ECD) of theLIV-1 gone product), which soluble form is useful as an antigen forraising anti-LIV-1 gene product antibodies that bind the extracellulardomain of full length LIV-1 gene product and inhibit its activation. Itis understood that LIV-1 gene product may also refer to the messengerRNA (mRNA) gene product and, where appropriate, the distinction betweenprotein and mRNA is made. A LIV-1 protein according to the invention isencoded by a nucleic acid of the invention comprising a sequence atleast 80% homologous to the DNA 164647 (SEQ ID NO:3 or its complement;FIG. 2A), preferably at least approximately 90% homologous, morepreferably at least approximately 95%, and most preferably at leastapproximately 97% homologous to SEQ ID NO:3 or its complement), or aportion thereof. A LIV-1 nucleic acid of the invention hybridizes understringent conditions to SEQ ID NO:3 or its complement) or a portionthereof. A LIV-1 protein of the invention is at least 80% homologous tothe amino acid sequence encoded by DNA 164647 (LIV-1 polypeptide SEQ IDNO:4; FIG. 2B), preferably at least approximately 90% homologous, morepreferably at least approximately 95%, and most preferably at leastapproximately 97% homologous to SEQ ID NO:4, or a fragment thereof.

[0047] The terms “anti-LIV-1 antibody,” “LIV-1 antibody,” andgrammatically analogous terms refer to an antibody which bindsspecifically to at least a portion of the extracellular domain of theLIV-1-164647 protein having a predicted amino acid sequence of SEQ IDNO:4 (the predicted full length amino acid sequence of LIV-1-164647gene). Preferably, the antibody binds to the extracellular domain ofLIV-1 gene product, more preferably binding to the same epitope asepitopes A, B, or C to which the monoclonal antibodies disclosed hereinbind. Even more preferably, the anti-LIV-1-164647 antibody binds to apolypeptide having at least 65% homology to a sequence from amino acid114 to and including amino acid 135 of SEQ ID NO:4. Preferably, ananti-LIV-1 antibody of the invention is human or humanized when theantibody is to be used to treat a human patient.

[0048] The antibody of the invention is preferably one which bindsspecifically to human LIV-1-164647, meaning that it does notsignificantly cross-react with other proteins. In such embodiments, theextent of binding of the antibody to proteins other than LIV-1 geneproduct will be less than about 10% as determined by fluorescenceactivated cell sorting (FACS) analysis or radioimmunoprecipitation(RIA).

[0049] The term “antibody” is used in the broadest sense andspecifically covers, for example, single anti-LIV-1 monoclonalantibodies (including agonist, antagonist, and neutralizing antibodies),anti-LIV-1 antibody compositions with polyepitopic specificity, singlechain anti-LIV-1 antibodies. The term “monoclonal antibody” as usedherein refers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts.

[0050] “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteinshaving the same structural characteristics. While antibodies exhibitbinding specificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kiln are, for example, producedat low levels by the lymph system and at increased levels by myelomas.The term “antibody” is used in the broadest sense and specificallycovers, without limitation, intact monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g. bispecific antibodies) formedfrom at least two intact antibodies, and antibody fragments so long asthey exhibit the desired biological activity.

[0051] “Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aliened with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

[0052] The term “variable” refers to the fact that certain portions ofthe variable domains differ extensively in sequence among antibodies andare used in the binding and specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed throughout the variable domains of antibodies. It isconcentrated in three segments called complementarity-determiningregions (CDRs) or hypervariable regions both in the light-chain and theheavy-chain variable domains. The more highly conserved portions ofvariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a -sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the -sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen-binding site of antibodies (see Kabat etal. NIH Publ. No.91-3242, Vol. 1 pages 647-669 (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

[0053] The term “hypervariable region” when used herein refers to theamino acid residues of an antibody which are responsible forantigen-binding. The hypervariable region comprises amino acid residuesfrom a “complementarity determining region” to “CDR” (i.e., residues inthe light chain variable domain and residues in the heavy chain variabledomain; Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institute of Health, Bethesda,Md. [1991]) and/or those residues from a “hypervariable loop” (i.e.,residues in the light chain variable domain and residues in the heavychain variable domain; Clothia and specificity, the monoclonalantibodies are advantageous in that they are synthesized by thehybridoma culture, uncontaminated by other immunoglobulins. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 [1975], or may be made byrecombinant DNA methods (see, e.g. U.S. Pat. No. 4,816,567). Themonoclonal antibodies may also be isolated from phage antibody librariesusing the techniques described in Clackson et al., Nature, 352:624-628[1991] and Marks et al., J. Mol. Biol. 222:581-597 (1991), for example.

[0054] The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; Morrison et al.,Proc. Natl. Acad. Sci. USA. 81:6851-6855 [1984]). Chimeric antibodies ofinterest herein include human constant region sequences together withantigen bindings regions of rodent (e.g. murine) origin, or “primatized”antibodies comprising variable domain antigen-binding sequences derivedfrom a non-human primate (e.g. Old World Monkey, Ape, macaque, etc.), orantigen binding regions derived from antibodies generated in othernon-human species that have been immunized with the antigen of interest.

[0055] “Humanized” forms of non-human (e.g., rodent) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity. In some instances, framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are foundneither in the recipient antibody nor in the donor antibody. Thesemodifications are made to further refine and maximize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see, Jones et al., Nature,311:522-525 (1986); Reichmann et al., Nature, 332:323-329 [1988]; andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0056] “Single-chain Fv” or “sFv” antibody fragments comprise the V_(H)and V_(L) domains of antibody, wherein these domains are present in asingle polypeptide chain. Preferably, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenhurg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

[0057] The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)−V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA. 90:6444-6448 (1993).

[0058] An “isolated” antibody is one which has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% (by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antihody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

[0059] The term “overexpression,” as used herein refers tooverexpression of a gene and/or its encoded protein in a cell, such as acancer cell. A cancer cell that “overexpresses” a protein is one thathas significantly higher levels of that protein compared to anoncancerous cell of the same tissue type. For example, according to thepresent invention, the overexpression of a protein LIV-1 protein may becaused by gene amplification or by increased transcription ortranslation.

[0060] Overexpression of a LIV-1 protein may be determined in adiagnostic or prognostic assay by evaluating increased levels of a LIV-1mRNA in a cell or tissue (e.g. via a quantitative PCR method) or bydetecting a LIV-1 protein present on the surface of a cell (e.g. via animmunohistochemistry assay). Alternatively, or additionally, one maymeasure levels of LIV-1-encoding nucleic acid in the cell, e.g. viafluorescent in situ hybridization (FISH; see WO98/45479 publishedOctober, 1998), southern blotting, or polymerase chain reaction (PCR)techniques, such as real time quantitative PCR (RT-PCR). One may alsostudy LIV-1 overexpression by measuring shed antigen (e.g., LIV-1extracellular domain) in a biological fluid such as serum by contactingthe fluid or other sample with an antibody that binds to a LIV-1 proteinor fragment thereof. Various in vitro and in vivo assays are availableto the skilled practitioner. For example, one may expose a fluid ortissue comprising a LIV-1 protein or fragment thereof, or cells withinthe body of the patient to an antibody which is optionally labeled witha detectable label, e.g. a radioactive isotope, and binding of theantibody to cells in a sample or the patient can be evaluated foroverexpression, e.g. by external scanning for radioactivity or byanalyzing a biopsy taken from a patient previously exposed to theantibody.

[0061] A cell that “overexpresses” LIV-1 has significantly higher thannormal LIV-1 nucleic acid levels compared to a noncancerous cell of thesame tissue type. Typically, the cell is a cancer cell, e.g. a breast,ovarian, prostate, stomach, endometrial, salivary gland, lung, kidney,colon, thyroid, pancreatic or bladder cell. The cell may also be a cellline such as SKBR3, BT474, Calu 3, MDA-MB-453, MDA-MB-361 or SKOV3.

[0062] Conversely, a cancer that is “not characterized by overexpressionof a LIV-1 protein or a LIV-1 gene is one which, in a diagnostic assay,does not express higher than normal levels of LIV-1 gene or LIV-1protein compared to a noncancerous cell of the same tissue type.

[0063] The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include highest cancer, prostatecancer, colon cancer, squamous cell cancer, small-cell lunge cancer,non-small cell lunar cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, colorectal cancer, endometrial carcinoma, salivarygland carcinoma kidney cancer, liver cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer.

[0064] The phrases “gene amplification” and “gene duplication” are usedinterchangeably and refer to a process by which multiple copies of agene or gene fragment are formed in a particular cell or cell line. Theduplicated region (a stretch of amplified DNA) is often referred to as“amplicon.” Usually, the amount of the messenger RNA (mRNA) produced,i.e., the level of gene expression, also increases in the proportion ofthe number of copies made of the particular gene expressed.

[0065] “Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

[0066] “Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of a disorder.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. In tumor (e.g., cancer) treatment, atherapeutic agent may directly decrease the pathology of tumor cells, orrender the tumor cells more susceptible to treatment by othertherapeutic agents, e.g., radiation and/or chemotherapy.

[0067] The “pathology” of cancer includes all phenomena that compromisethe well-being of the patient. This includes, without limitation,abnormal or uncontrollable cell growth, metastasis, interference withthe normal functioning of neighboring cells, release of cytokines orother secretory products at abnormal levels, suppression or aggravationof inflammatory or immunological response, etc.

[0068] “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, horses, cats, cattle, pigs,sheep, etc. Preferably, the mammal is human.

[0069] “Carriers” as used herein include pharmaceutically acceptablecarriers, excipients, or stabilizers which are nontoxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.Often the physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids,antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming, counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0070] Administration “in combination with” one or more furthertherapeutic agents includes simultaneous (concurrent) and consecutiveadministration in any order.

[0071] The term “cytotoxic agent” as used herein refers to a substancethat inhibits or prevents the function of cells and/or causesdestruction of cells. The term is intended to include radioactiveisotopes (e.g., I¹³¹, I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, andtoxins such as enzymatically active toxins of bacterial, fungal, plantor animal origin, or fragments thereof.

[0072] A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeadriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosinearabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin,taxoids, e.g., paclitaxel (Taxol, Bristol-Myers Squibb Oncology,Princeton, N.J.), and doxetaxel (Taxotere, Rhône-Poulenc Rorer, Antony,Rnace), toxotere, methotrexate, cisplatin, melphalan, vinblastine,bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone,vincristine, vinorelbine, carboplatin, teniposide, daunomycin,carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (seeU.S. Pat. No. 4,675,187), 5-FU, 6-thioguanine, 6-mercaptopurine,actinomycin D, VP-16, chlorambucil, melphalan, and other relatednitrogen mustards. Also included in this definition are hormonal agentsthat act to regulate or inhibit hormone action on tumors such astamoxifen and onapristone.

[0073] A “growth inhibitory agent” when used herein refers to a compoundor composition which inhibits growth of a cell, especially cancer celloverexpressing any of the genes identified herein, either in vitro or invivo. Thus, the growth inhibitory agent is one which significantlyreduces the percentage of cells overexpressing such genes in S phase.Examples of growth inhibitory agents include agents that block cellcycle progression (at a place other than S phase), such as agents thatinduce G1 arrest and M-phase arrest. Classical M-phase blockers includethe vincas (vincristine and vinblastine), taxol, and topo II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest GI also spill over into S-phase arrest, forexample. DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogens, and antineoplastic drugs” by Murakami et al., (WBSaunders; Philadelphia, 1995), especially p. 13.

[0074] “Doxorubicin” is an athracycline antibiotic. The full chemicalname of doxorubicin is(85-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

[0075] The term “cytokine” is a generic term for proteins released byone cell population which act on another cell as intercellularmediators. Examples of such cytokines are lymphokines, monokines, andtraditional polypeptide hormones. Included among the cytokines aregrowth hormone such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associatcd peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α orTNF-β; and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

[0076] The term “prodrug” as used in this application refers to aprecursor or derivative form of a pharmaceutically active substance thatis less cytotoxic to tumor cells compared to the parent drug and iscapable of being enzymatically activated or converted into the moreactive parent form. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy”Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast(1986) and Stella et al., “Prodrugs: A Chemical Approach to TargetedDrug Delivery.” Directed Drug Delivery, Borchardt et al., (ed.), pp.247-267. Humana Press (1985). The prodrugs of this invention include,but are not limited to, phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs. D-amino acid-modified prodrugs,glycosylated prodrugs, β-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs. 5-fluorocytosing andother 5-fluorouridine prodrugs which can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrug form for use in this invention include, butare not limited to, those chemotherapeutic agents described above.

[0077] An “effective amount” of a polypeptide disclosed herein or anantagonist thereof, in reference to inhibition of neoplastic cellgrowth, tumor growth or cancer cell growth, is an amount capable ofinhibiting to some extent, the growth of target cells. The term includesan amount capable of invoking a growth inhibitory, cytostatic and/orcytotoxic effect and/or apoptosis of the target cells. An “effectiveamount” of a LIV-1 polypeptide antagonist for purposes of inhibitingneoplastic cell growth, tumor growth or cancer cell (growth, may bedetermined empirically and in a routine manner.

[0078] A “therapeutically effective amount”, in reference to thetreatment of tumor, refers to an amount capable of invoking one or moreof the following effects: (1) inhibition, to some extent, of tumorgrowth, including, slowing down and complete growth arrest; (2)reduction in the number of tumor cells; (3) reduction in tumor size; (4)inhibition (i.e., reduction, slowing down or complete stopping) of tumorcell infiltration into peripheral organs; (5) inhibition (i.e.,reduction, slowing down or complete stopping) of metastasis; (6)enhancement of anti-tumor immune response, which may, but does not haveto, result in the regression or rejection of the tumor; and/or (7)relief, to some extent, of one or more symptoms associated with thedisorder. A “therapeutically effective amount” of a LIV-1 polypeptideantagonist for purposes of treatment of tumor may be determinedempirically and in a routine manner.

[0079] A “growth inhibitory amount” of a LIV-1 antagonist is an amountcapable of inhibiting the growth of a cell, especially tumor, e.g.,cancer cell, either in vitro or in vivo. A “growth inhibitory amount” ofa LIV-1 antagonist for purposes of inhibiting neoplastic cell growth maybe determined empirically and in a routine manner.

[0080] A “cytotoxic amount” of a LIV-1 antagonist is an amount capableof causing the destruction of a cell, especially tumor, e.g., cancercell, either in vitro or in vivo. A “cytotoxic amount” of a LIV-1antagonist for purposes of inhibiting neoplastic cell growth may bedetermined empirically and in a routine manner.

[0081] “Percent (%) amino acid sequence homology or identity” withrespect to the LIV-1 polypeptide sequences identified herein is definedas the percentage of amino acid residues in a candidate sequence thatare identical with the amino acid residues in a LIV-1 sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2, whereinthe complete source code for the ALIGN-2 program was authored byGenentech, Inc., and the source code shown in FIGS. 20A-Q has been filedwith user documentation in the U.S. Copyright Office. Washington D.C.,20559, where it is registered under U.S. Copyright Registration No.TXU510087, and is provided in Table 1. The ALIGN-2 program is publiclyavailable through Genentech, Inc. South San Francisco, Calif. TheALIGN-2 program should be compiled for use on a UNIX operating system,preferably digital UNIX V4.0D. All sequence comparison parameters areset by the ALIGN-2 program and do not vary.

[0082] For purposes herein, the % amino acid sequence identity of agiven amino acid sequence A to, with, or against a given amino acidsequence B (which can alternatively be phrased as a given amino acidsequence A that has or comprises a certain % amino acid sequenceidentity to, with or against a given amino acid sequence B) iscalculated as follows:

100 times the fraction X/Y

[0083] where X is the number of amino acid residues scored as identicalmatches by the sequence alignment program ALIGN-2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

[0084] Unless specifically stated otherwise, all % amino acid sequencehomology or identity values used herein arc obtained as described aboveusing the ALIGN-2 sequence comparison computer program. However, % aminoacid sequence identity may also be determined using the sequencecomparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res.,25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may bedownloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses severalsearch parameters, wherein all of those search parameters are set todefault values including, for example, unmask=yes, strand=all, expectedoccurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

[0085] In situations where NCBI-BLAST2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

[0086] where X is the number of amino acid residues scored as identicalmatches by the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

[0087] In addition, % amino acid sequence identity may also bedetermined using the WU-BLAST-2 computer program (Altschul et al.,Methods in Enzymology, 266:460-480 (1996)). Most of the WU-BLAST-2search parameters are set to the default values. Those not set todefault values, i.e., the adjustable parameters, are set with thefollowing values: overlap span=1, overlap fraction=0.125, word threshold(T)=11, and scoring matrix=BLOSUM62. For purposes herein, a % amino acidsequence identity value is determined by dividing (a) the number ofmatching identical amino acids residues between the amino acid sequenceof the LIV-1 polypeptide of interest having a sequence derived from thenative LIV-1 polypeptide encoded by DNA 164647 and the comparison aminoacid sequence of interest (i.e., the sequence against which the LIV-1polypeptide of interest is being compared which may be a LIV-1 variantpolypeptide) as determined by WU BLAST-2 by (b) the total number ofamino acid residues of the LIV-1 polypeptide of interest. For example,in the statement “a polypeptide comprising an amino acid sequence Awhich has or having at least 80% amino acid sequence identity to theamino acid sequence B”, the amino acid sequence A is the comparisonamino acid sequence of interest and the amino acid sequence B is theamino acid sequence of the LIV-1 polypeptide of interest.

[0088] “Percent (%) nucleic acid sequence homology or identity” withrespect to the LIV-1 polypeptide-encoding nucleic acid sequencesidentified herein is defined as the percentage of nucleotides in acandidate sequence that are identical with the nucleotides in a LIV-1polypeptide-encoding nucleic acid sequence, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity. Alignment for purposes of determining percent nucleicacid sequence identity can be achieved in various ways that are withinthe skill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full-length of the sequences being compared.For purposes herein, however, % nucleic acid sequence identity valuesare obtained as described below by using the sequence comparisoncomputer program ALIGN-2, wherein the complete source code for theALIGN-2 program is has been filed with user documentation in the U.S.Copyright Office, Washington D.C., 20559, where it is registered underU.S. Copyright Registration No. TXU510087 and is provided herein inTable 1 as source code. The ALIGN-2 program is publicly availablethrough Genentech, Inc., South San Francisco, Calif. The ALIGN-2 programshould be compiled for use on a UNIX operating system, preferablydigital UNIX V4.0D. All sequence comparison parameters are set by theALIGN-2 program and do not vary

[0089] For purposes herein, the % nucleic acid sequence identity of agiven nucleic acid sequence C to, with, or against a given nucleic acidsequence D (which can alternatively be phrased as a given nucleic acidsequence C that has or comprises a certain % nucleic acid sequenceidentity to with, or against a given nucleic acid sequence D) iscalculated as follows:

100 times the fraction W/Z

[0090] where W is the number of nucleotides scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofC and D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

[0091] Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described above using theALIGN-2 sequence comparison computer program. However, % nucleic acidsequence identity may also be determined using the sequence comparisonprogram NCBI-BLAST2 (Altschul et al., Nucleic Acids Res., 25:3389-3402(1997)). The NCBI-BLAST2 sequence comparison program may be downloadedfrom http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several searchparameters, wherein all of those search parameters are set to defaultvalues including, for example, unmask=yes, strand=all, expectedoccurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

[0092] In situations where NCBI-BLAST2 is employed for sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

100 times the fraction W/Z

[0093] where W is the number of nucleotides scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of C and D, and where Z is the total number of nucleotides inD. It will be appreciated that where the length of nucleic acid sequenceC is not equal to the length of nucleic acid sequence D, the % nucleicacid sequence identity of C to D will not equal the % nucleic acidsequence identity of D to C.

[0094] In addition, % nucleic acid sequence identity values may also begenerated using the WU-BLAST-2 computer program (Altschul et al.,Methods in Enzymology, 266:460-480 (1996)). Most of the WU-BLAST-2search parameters are set to the default values. Those not set todefault values, i.e., the adjustable parameters, are set with thefollowing values: overlap span=1, overlap fraction=0.125, word threshold(T)=11, and scoring matrix=BLOSUM62. For purposes herein, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of theLIV-1 polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence LIV-1 polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the LIV-1 polypeptide-encoding nucleic acidmolecule of interest is being compared which may be a variant LIV-1polynucleotide) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the LIV-1 polypeptide-encoding nucleic acid molecule ofinterest. For example, in the statement “an isolated nucleic acidmolecule comprising a nucleic acid sequence A which has or having atleast 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the LIV-1 polypeptide-encoding nucleic acid molecule of interest.

[0095] The term “positives”, in the context of the amino acid sequenceidentity comparisons performed as described above, includes amino acidresidues in the sequences compared that are not only identical, but alsothose that have similar properties. Amino acid residues that score apositive value to an amino acid residue of interest are those that areeither identical to the amino acid residue of interest or are apreferred substitution (as defined in Table 2 below) of the amino acidresidue of interest. TABLE 2 Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln;asn lys Asn (N) gln: his; lys; arg gln Asp (D) glu glu Cys (C) ser serGln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln;lys; arg arg Ile (I) leu; val; met; ala; phe; leu norleucine Leu (L)norleucine: ile: val; ile met; ala; phe Lys (K) arg; gln; asn are Met(M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) alaala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp;phe; thr; ser phe Val (V) ile; leu; met; phe; leu ala; norleucine

[0096] Substantial modifications in function or immunological identityof the polypeptide are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

[0097] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0098] (2) neutral hydrophilic: cys, ser, thr;

[0099] (3) acidic: asp, glu;

[0100] (4) basic: asn, gln, his, lys, arg;

[0101] (5) residues that influence chain orientation: gly, pro; and

[0102] (6) aromatic: trp, tyr, phe.

[0103] Non-conservative substitutions will entail exchanging a member ofone of these classes for another class. Such substituted residues alsomay be introduced into the conservative substitution sites or, morepreferably, into the remaining (non-conserved) sites.

[0104] The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986), Zoller et al., Nucl. Acids Res. 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performed.

[0105] For purposes herein, the % value of positives of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % positives to, with, or against a givenamino acid sequence B) is calculated as follows:

100 times the fraction X/Y

[0106] where X is the number of amino acid residues scoring a positivevalue as defined above by the sequence alignment program ALIGN-2 in thatprogram's alignment of A and B, and where Y is the total number of aminoacid residues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acid sequenceB, the % positives of A to B will not equal the % positives of B to A.

[0107] “Isolated.” when used to describe the various polypeptidesdisclosed herein, means polypeptide that has been identified andseparated and/or recovered from a component of its natural environment.Preferably, the isolated polypeptide is free of association with allcomponents with which it is naturally associated. Contaminant componentsof its natural environment are materials that would typically interferewith diagnostic or therapeutic uses for the polypeptide, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In preferred embodiments, the polypeptide will be purified (1) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (2)to homogeneity by SDS-PAGE under non-reducing or reducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated polypeptideincludes polypeptide in situ within recombinant cells, since at leastone component of the LIV-1 natural environment will not be present.Ordinarily, however, isolated polypeptide will be prepared by at leastone purification step.

[0108] An “isolated” nucleic acid molecule encoding a LIV-1 polypeptideor an “isolated” nucleic acid encoding an anti-LIV-1 antibody, is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the LIV-1-encoding nucleic acid or theanti-LIV-1-encoding nucleic acid. Preferably, the isolated nucleic acidis free of association with all components with which it is naturallyassociated. An isolated LIV-1-encoding nucleic acid molecule or ananti-LIV-1-encoding nucleic acid molecule is other than in the form orsetting in which it is found in nature.

[0109] The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

[0110] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice.

[0111] “Stringency” of hybridization reactions is readily determinableby one of ordinary skill in the art, and generally is an empiricalcalculation dependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers. (1995).

[0112] “Stringent conditions” or “high stringency conditions”, asdefined herein, may be identified by those that: (1) employ low ionicstrength and high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate). 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

[0113] “Moderately stringent conditions” may be identified as describedby Sambrook et al., Molecular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Press, 1989, and include the use of washing solutionand hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 35-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

[0114] “Active” or “activity” for the purposes herein refers to form(s)of LIV-1 polypeptides which retain a biological and/or an immunologicalactivity/property of a native or naturally-occurring LIV-1 polypeptide,wherein “biological” activity refers to a function (either inhibitory orstimulatory) caused by a native or naturally-occurring LIV-1 polypeptideother than the ability to induce the production of an antibody againstan antigenic epitope possessed by a a native or naturally-occurringLIV-1 polypeptide and an “immunological” activity refers to the abilityto induce the production of an antibody against an antigenic epitopepossessed by a native or naturally-occurring LIV-1 polypeptide.

[0115] “Biological activity” in the context of an antibody or anotherantagonist molecule that can be identified by the screening assaysdisclosed herein (e.g., an organic or inorganic small molecule, peptide,etc.) is used to refer to the ability of such molecules to bind orcomplex with the polypeptides encoded by the amplified genes identifiedherein, or otherwise interfere with the interaction of the encodedpolypeptides with other cellular proteins or otherwise interfere withthe transcription or translation of a LIV-1 polypeptide. A preferredbiological activity is growth inhibition of a target tumor cell. Anotherpreferred biological activity is cytotoxic activity resulting in thedeath of the target tumor cell.

[0116] The term “biological activity” in the context of a LIV-1polypeptide means the ability of a LIV-1 polypeptide to induceneoplastic cell growth or uncontrolled cell growth or to act as anindication of a particular form of neoplasm that is particularlymetastatic.

[0117] The phrase “immunological activity” means immunologicalcross-reactivity with at least one epitope of a LIV-1 polypeptide.

[0118] “Immunological cross-reactivity” as used herein means that thecandidate polypeptide is capable of competitively inhibiting thequalitative biological activity of a LIV-1 polypeptide having thisactivity with polyclonal antisera raised against the known active LIV-1polypeptide. Such antisera are prepared in conventional fashion byinjecting goats or rabbits, for example, subcutaneously with the knownactive analogue in complete Freund's adjuvant, followed by boosterintraperitoneal or subcutaneous injection in incomplete Freunds. Theimmunological cross-reactivity preferably is “specific”, which meansthat the binding affinity of the immunologically cross-reactive molecule(e.g. antibody) identified, to the corresponding LIV-1 polypeptide issignificantly higher (preferably at least about 2-times, more preferablyat least about 4-times, even more preferably at least about 8-times,most preferably at least about 10-times higher) than the bindingaffinity of that molecule to any other known native polypeptide.

[0119] The term “antagonist” is used in the broadest sense, and includesany molecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native LIV-1 polypeptide disclosed herein orthe transcription or translation thereof. Suitable antagonist moleculesspecifically include antagonist antibodies or antibody fragments,fragments, peptides, small organic molecules, anti-sense nucleic acids,etc. Included are methods for identifying antagonists of a LIV-1polypeptide with a candidate antagonist molecule and measuring adetectable chance in one or more biological activities normallyassociated with the LIV-1 polypeptide.

[0120] A “small molecule” is defined herein to have a molecular weightbelow about 500 Daltons.

[0121] The word “label” when used herein refers to a detectable compoundor composition which is conjugated directly or indirectly to theantibody so as to generate a “labeled” antibody. The label may bedetectable by itself (e.g., radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable.Radionuclides that can serve as detectable labels include, for example,I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, andPd-109.

[0122] By “solid phase” is meant a non-aqueous matrix to which theantibody of the present invention can adhere. Examples of solid phasesencompassed herein include those formed partially or entirely of glass(e.g., controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate: in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

[0123] A “liposome” is a small vesicle composed of various types oflipids, phospholipids and/or surfactant which is useful for delivery ofa drug (such as a LIV-1 polypeptide or antibody thereto and, optionally,a chemotherapeutic agent) to a mammal. The components of the liposomeare commonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes.

[0124] As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin,” for example a receptor, ligand, or enzyme) withthe effector functions of immunoglobulin constant domains. Structurally,the immunoadhesins comprise a fusion of the adhesin amino acid sequencewith the desired binding specificity which is other than the antigenrecognition and binding site (antigen combining site) of an antibody(i.e., is “heterologrous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontinuous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes. IgA (including IgA-1 and IgA-2), IgE,IgD or IgM, and any subclass or isotype thereof.

[0125] The terms “HER2”, “ErbB2” “c-Erb-B2” are used interchangeably.Unless indicated otherwise, the terms “ErbB2” “c-Erb-B2” and “HER2” whenused herein refer to the human protein, and “erbB2,” “c-erb-B2,” and“her2” refer to human gene. The human erbB2 gene and ErbB2 protein are,for example, described in Semba et al., PNAS (USA) 82:6497-6501 (1985)and Yamamoto et al., Nature 319:230-234 (1986) (Genebank accessionnumber X03363). ErbB2 comprises four domains (Domains 1-4).

[0126] The “epitope 4D5” is the region in the extracellular domain ofErbB2 to which the antibody 4D5 (ATCC CRL 10463) binds. This epitope isclose to the transmembrane region of ErbB2. To screen for antibodieswhich bind to the 4D5 epitope, a routine cross-blocking assay such asthat described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed to assess whether theantibody binds to the 4D5 epitope of ErbB2 (i.e. any one or moreresidues in the region from about residue 529, e.g. about residue 561 toabout residue 625, inclusive).

[0127] The “epitope 3H4” is the region in the extracellular domain ofErbB2 to which the antibody 3H4 binds. This epitope includes residuesfrom about 541 to about 599, inclusive, in the amino acid sequence ofErbB2 extracellular domain.

[0128] The “epitope 7C2/7F3” is the region at the N terminus of theextracellular domain of ErbB2 to which the 7C2 and/or 7F3 antibodies(ATCC HB-12215 and ATCC HB-12216, respectively) bind. To screen forantibodies which bind to the 7C2/7F3 epitope, a routine cross-blockingassay such as that described in Antibodies, A Laboratory Manual, ColdSpring Harbor Laboratory, Ed Harlow and David Lane (1988), can beperformed. Alternatively, epitope mapping can be performed to establishwhether the antibody binds to the 7C2/7F3 epitope on ErbB2.

[0129] The term “induces cell death” or “capable of inducing cell death”refers to the ability of the anti-LIV-1 gene product antibody, alone orin co-treatment with a chemotherapeutic agent, to make a viable cellbecome nonviable. The “cell” here is one which expresses the LIV-1 geneproduct, especially where the cell overexpresses the LIV-1 gene product.A cell which “overexpresses” LIV-1 has significantly higher than normalLIV-1 mRNA and/or LIV-1 protein levels compared to a noncancerous cellof the same tissue type. A cell to be treated by the method of theinvention does not also overexpress ErbB2 (i.e. the cell expresses ErbB2at a level that is approximately the same or less than a normal,non-cancerous cell of the same cell or tissue type). Preferably, thecell is a cancer cell, e.g. a breast, lung, or prostate cell, In vitro,the cell may be from a cell line transformed with LIV-1 DNA, preferablyDNA 164647, to express LIV-1 on the cell surface. Cell death in vitromay be determined in the absence of complement and immune effector cellsto distinguish cell death induced by antibody dependent cellularcytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Thus,the assay for cell death may be performed using heat inactivated serum(i.e. in the absence of complement) and in the absence of immuneeffector cells. To determine whether the antibody is able to induce celldeath, loss of membrane integrity as evaluated by uptake of propidiumiodide (PI), trypan blue (see Moore et al., Cytotechnology 17:1-11[1995]) or 7AAD can be assessed relative to untreated cells. Preferredcell death-inducing antibodies are those which induce PI uptake in the“PI uptake assay in LIV-1 expressing cells”.

[0130] The phrase “induces apoptosis” or “capable of inducing apoptosis”refers to the ability of the antibody to induce programmed cell death asdetermined by binding of annexin V, fragmentation of DNA, cellshrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/orformation of membrane vesicles (called apoptotic bodies). The cell isone which overexpresses the LIV-1 gene product. Preferably the “cell” isa tumor cell, e.g. a breast, lung, or prostate cell. In vitro, the cellmay be from a cell line transformed with LIV-1 DNA, such as DNA 164647.Various methods are available for evaluating the cellular eventsassociated with apoptosis. For example, phosphatidyl serine (PS)translocation can be measured by annexin binding; DNA fragmentation canbe evaluated through DNA laddering as disclosed in the example herein;and nuclear/chromatin condensation along with DNA fragmentation can beevaluated by any increase in hypodiploid cells. Preferably, the antibodywhich induces apoptosis is one which results in about 2 to 50 fold,preferably about 5 to 50 fold, and most preferably about 10 to 50 foldinduction of annexin binding relative to untreated cell in an “annexinbinding assay using cells” (see below).

[0131] As used herein, the term “salvage receptor binding epitope”refers to an epitope of the Fc region of an IgG molecule (e.g., IgG₁,IgG₂, IgG₃, or IgG₄) that is responsible for increasing the in vitroserum half-life of the IgG molecule.

[0132] “Treatment” refers to both therapeutic treatment and prophylacticor preventative measures. Those in need of treatment include thosealready with the disorder as well as those in which the disorder is tobe prevented.

[0133] A “disorder” is any condition that would benefit from treatmentwith the anti-LIV-1 gene product antibody. This includes chronic andacute disorders or diseases including those pathological conditionswhich predispose the mammal to the disorder in question. Non-limitingexamples of disorders to be treated herein include benign and malignanttumors of breast, lung, and prostate tissue.

[0134] The term “package insert” is used to refer to instructionscustomarily included in commercial packages of therapeutic products,that contain information about the indications, usage, dosage,administration, contraindications and/or warnings concerning the use ofsuch therapeutic products.

[0135] The term “serum concentration,” “serum drug concentration,” orserum anti-LIV-1 “antibody concentration” refers to the concentration ofa drug in the blood serum of an animal or human patient being treatedwith the drugs. Serum concentration of antibody, for example, ispreferably determined by immunoassay. Preferably, the immunoassay isELISA according to the procedure disclosed herein.

[0136] The term “peak serum concentration” refers to the maximal serumdrug concentration shortly after delivery of the drug into the animal orhuman patient, after the drug has been distributed throughout the bloodsystem, but before significant tissue distribution, metabolism orexcretion of drug by the body has occurred.

[0137] The term “trough serum concentration” refers to the serum drugconcentration at a time after delivery of a previous dose andimmediately prior to delivery of the next subsequent dose of drug in aseries of doses. Generally, the trough serum concentration is a minimumsustained efficacious drug concentration in the series of drugadministrations. Also, the trough serum concentration is frequentlytargeted as a minimum serum concentration for efficacy because itrepresents the serum concentration at which another dose of drug is tobe administered as part of the treatment regimen. If the delivery ofdrug is by intravenous administration, the trough serum concentration ismost preferably attained within 1 day of a front loading initial drugdelivery. It the delivery of drug is by subcutaneous administration, thepeak serum concentration is preferably attained in 3 days or less.According to the invention, the trough serum concentration is preferablyattained in 4 weeks or less, preferably 3 weeks or less, more preferably2 weeks or less, most preferably in 1 week or less, including 1 day orless using any of the drug delivery methods disclosed herein.

[0138] The term “intravenous infusion” refers to introduction of a druginto the vein of an animal or human patient over a period of timegreater than approximately 5 minutes, preferably between approximately30 to 90 minutes, although, according to the invention, intravenousinfusion is alternatively administered for 10 hours or less.

[0139] The term “intravenous bolus” or “intravenous push” refers to drugadministration into a vein of an animal or human such that the bodyreceives the drug in approximately 15 minutes or less, preferably 5minutes or less.

[0140] The term “subcutaneous administration” refers to introduction ofa drug under the skin of an animal or human patient, preferable within apocket between the skin and underlying tissue, by relatively slow,sustained delivery from a drug receptacle. The pocket may be created bypinching or drawing the skin up and away from underlying tissue.

[0141] The term “subcutaneous infusion” refers to introduction of a drugunder the skin of an animal or human patient, preferably within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle for a period of time including, but notlimited to, 30 minutes or less, or 90 minutes or less. Optionally, theinfusion may be made by subcutaneous implantation of a drug deliver,pump implanted under the skin of the animal or human patient, whereinthe pump delivers a predetermined amount of drug for a predeterminedperiod of time, such as 30 minutes, 90 minutes, or a time periodspanning the length of the treatment regimen.

[0142] The term “subcutaneous bolus” refers to drug administrationbeneath the skin of an animal or human patient, where bolus drugdelivery is preferably less than approximately 15 minutes, morepreferably less than 5 minutes, and most preferably less than 60seconds. Administration is preferably within a pocket between the skinand underlying tissue, where the pocket is created, for example, bypinching or drawing the skin up and away from underlying tissue.

[0143] The term “front loading,” when referring to drug administrationis meant to describe an initially higher dose followed by the same orlower doses at intervals. The initial higher dose or doses are meant tomore rapidly increase the animal or human patient's serum drugconcentration to an efficacious target serum concentration.

[0144] Published information related to LIV-1 gene expression and geneproduct includes the following issued patents and publishedapplications: Manning, D. L. et al., U.S. Pat. No. 5,693,465, issuedDec. 2, 1997; Manning, D. L. et al., European J. Cancer29A(10):1462-1468 [1993]; Manning, D. L. et al., European J. Cancer,30A(5):675-678 [1994]; Manning. D. L. et al., Acta Oncologica34(5):641-646 [1995]; McClelland, R. A. et al., Breast Cancer Res. &Treatment 41(1):31-41 [1996]; Knowlden, J. M. et al. Clin. Cancer Res.3(11):2165-2172 [1997]; and McClelland, R. A. et al., British J. Cancer77(10):1653-1656 [1998].

[0145] Published information related to anti-ErbB2 antibody includes thefollowing issued patents and published applications: PCT/US89/00051,published Jan. 5, 1989, PCT/US90/02697, published May 18, 1990, EU0474727, issued Jul. 23, 1997, DE 69031120.6, issued Jul. 23, 1997,PCT/US97/18385, published Oct. 9, 1997, SA 97/9185, issued Oct. 14,1997, U.S. Pat. No. 5,677,171, issued Oct. 14, 1997, U.S. Pat. No.5,720,937, issued Feb. 24, 1998. U.S. Pat. No. 5,720,954, issued Feb.24, 1998, U.S. Pat. No. 5,725,856, issued Mar. 10, 1998, U.S. Pat. No.5,770,195, issued Jun. 23, 1998, U.S. Pat. No. 5,772,997, issued Jun.30, 1998, PCT/US98/2626, published Dec. 10, 1998, and PCT/US99/06673,published Mar. 26, 1999, each of which patents and publications isherein incorporated by reference in its entirety.

[0146] The following examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0147] All patent and literature references cited in the presentspecification are hereby incorporated by reference in their entirety.

EXAMPLES

[0148] Commercially available reagents referred to in the examples wereused according to manufacturer's instructions unless otherwiseindicated. The source of those cells identified in the followingexamples, and throughout the specification, by ATCC accession numbers isthe American Type Culture Collection, Manassas, Va. Unless otherwisenoted, the present invention uses standard procedures of recombinant DNAtechnology, such as those described herein and in the followingtextbooks: Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press N.Y. 1989; Ausubel et al., Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y. 1989; Innis et al., PCR Protocols: A Guide to Methods andApplications, Academic Press, inc., N.Y. 1990; Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, 1988; Gait, M. J. Oligonucleotide Synthesis, IRL Press, Oxford,1984, R. I. Freshney, Animal Cell Culture, 1987 Coligan et al., CurrentProtocols in Immunology, 1991.

Example 1 LIV-1 Expression in Tumor Cells Examined by MicroarrayAnalysis

[0149] A form of breast cancer in which the cells overexpress the LIV-1gene product (e.g. LIV-1-164647 mRNA) but do not overexpress ErbB2 hasbeen discovered and is uniquely disclosed herein. Detection of the tumortype was made using microarray technology. Using nucleic acidmicroarrays, test and control mRNA samples from test and control tissuesamples are reverse transcribed and labeled to generate cDNA probes. ThecDNA probes are then hybridized to an array of nucleic acids immobilizedon a solid support. The array is configured such that the sequence andposition of each member of the array is known. Hybridization of alabeled probe with a particular array member indicates that the samplefrom which the probe was derived expresses that gene. If thehybridization signal of a probe from a test (disease tissue) sample isgreater than hybridization signal of a probe from a control (normaltissue) sample, the gene or genes overexpressed in the disease tissueare identified. The implication of this result is that an overexpressedprotein in a diseased tissue is useful not only as a diagnostic markerfor the presence of the disease condition, but also as a therapeutictarget for treatment of the disease condition.

[0150] Tumor cells were crossly dissected from surrounding,non-cancerous cells in breast tumor tissue. Hematoxylin and eosinstaining of the cells confirmed that the excised cells were from tumor.The mRNA of the tumor cells (reflecting cell-specific expression of avariety of genes) was converted to cDNA by RT/PCR methodology, labeledwith fluorescent tags, and allowed to hybridize to the ESTs arrayed on aclass slide. An imaging device detected and measured the fluorescence ofeach sample on the slide, where fluorescence represents a labeledmessenger from the test cells identifiable due to its hybridization witha known nucleic acid sequence (an EST) at a known position on the slide.Relative fluorescence indicated relative activity of a gene, with strongflourescence indicating an active gene expressing a relative largeamount of messenger. Little or no flourescence indicated that no labeledmessenger hybridized to the ESTs. Detection of fluorescently labeledprobes hybridized to sequences on the microarray slide is described, forexample, in U.S. Pat. No. 5,143,854, herein incorporated by reference.

[0151] It was found that cDNA of the preparation hybridized to apublicly available EST sequence (accession no. H29315 (from theWashington University-Merck EST Project, authors Hillier. L. et al., SEQID NO:5, purchased from Research Genetics (Alabama, USA) in a patternsuggesting overexpression in breast tumor tissue that did not alsooverexpress ErbB2. The cDNA was sequenced and disclosed herein asLIV-1-DNA 164647. In situ hybridization of a radioactively labeledLIV-1-164647 probe to a tissue microarray of tumor tissues furtherindicated LIV-1 expression in tumors from breast, lung, prostate, colon,endometrial, ovarian, and transitional carcinomas, and melanoma tissuesamples.

[0152] It was further discovered that breast tumor cells whichoverexpressed LIV-1 did not overexpress ErbB2 relative to a normal cellof the same type (where a normal cell refers to a cell that is notcancerous and free of any other disease in which LIV-1 may beoverexpressed). The very low expression of LIV-1 in normal tissuesrelative to the overexpression of LIV-1 in breast tumor cells makesLIV-1 a desirable target for a therapeutic antibody.

[0153] For example, in breast tumors, strong fluorescent detection ofLIV-1 occurred with low detection of ErbB2 expression, or alternativelystrong flourescent detection of ErbB2 was observed in the absence ofLIV-1 mRNA expression. Of 17 samples, each from a different breast tumorof variable tumor cell content, 6 samples showed moderate to strongLIV-1 expression, and 6 showed moderate or strong ErbB2 expression.There was an overlap in one tumor which showed moderate ErbB2 and LIV-1expression. In another tumor it appeared that strong detection of ErbB2expression was accompanied by very weak detection of LIV-1 expression.Cell lines expressing endogenous or exogenous LIV l protein (preferablyLIV-1-164647), but not overexpressing ErbB2, are thus useful for testingthe detection or LIV-1 expression relative to ErbB2 expression. Suchcells are also useful to test anti-LIV-1 antibodies for binding toLIV-1-164647 expressing cells and their affects on cell growth.

[0154] Regions of LIV-1-164647 in the ECD and in the C-terminus werefound to be unique as compared to the LIV-1 sequence of Green et al.,(see FIG. 4 comparing SEQ ID NO:4 and GenBank Accession No. AAA96258).The region spanned amino acids 126 to and including amino acid 132 ofSEQ ID NO:4 (SEQ ID NO:17) and amino acids 743 to 755 (SEQ ID NO:18). Alarder histidine-rich region from amino acid 114 to and including aminoacid 135 (SEQ ID NO:19) is also encompassed by the invention. Thus,according to the presently disclosed invention, the nucleic acid of theinvention comprises a sequence having at least 65%, preferably at least75%, more preferably at least 85%, still more preferably at least 90%,and most preferably at least 96% homologous to a sequence fromapproximately nucleotide 412 to and including nucleotide 477 of SEQ IDNO:3, or its complementary sequence. Preferably, the nucleic acid of theinvention comprises a sequence from nucleotide 443 to and includingnucleic acid 446 of SEQ ID NO:3. The present invention further includesan isolated polypeptide comprising an amino acid sequence having atleast 65%, preferably at least 75%, more preferably at least 85%, stillmore preferably at least 90%, and most preferably at least 96% homologyto a sequence from approximately amino acid 114 to and including aminoacid 135 of SEQ ID NO:4. Preferably, the isolated polypeptide of theinvention comprises an amino acid sequence from amino acid 126 to andincluding amino acid 132 of SEQ ID NO:4.

Example 2 Preparation of the LIV-1 Polypeptides

[0155] A previously unknown LIV-1 gene and its encoded protein areuniquely disclosed herein. The nucleic acid sequence of LIV-1 (DNA164647; SEQ ID NO:3 and its complement) and amino acid sequence of LIV-1encoded therein (SEQ ID NO:4) are uniquely disclosed herein. Thisexample describes the preparation and isolation of the presentlydisclosed LIV-1 protein encoded by DNA 164647. Subsequent examplesdescribe the tissue expression profile of LIV-1 and its partial exposureat the cell surface. The LIV-1 gene is disclosed herein to be expressedin various tumor tissues including breast, lung, prostate, and colon.Cell surface expression of this tumor-associated protein makes LIV-1 auseful target for cancer detection and treatment.

[0156] The LIV-1 described by DNA 164647 has a unique nucleic acidsequence and unique amino acid sequence. The presently disclosed LIV-1differs in nucleic acid sequence and amino acid sequence from anotherform of LIV-1 protein previously described by Green et al., (see Green,C. et al., direct submission, GenBank Accession Nos. U41060 (nucleicacid sequence) and AAA96258 (amino acid sequence)). The nucleic acidsequence of AAA96258 LIV-1 (SEQ ID NO:1 (coding sequence)) is shown inFIG. 1A. The predicted amino acid sequence (AAA96258; SEQ ID NO:2)encoded by nucleic acid sequence U41061 (SEQ ID NO: 1) is shown in FIG.1B. Included in the diagram are predictions for sequences encoding asignal sequence, extracellular domain, and transmembrane domain.Significantly, the presently disclosed LIV-1 (DNA 164647) differs inpredicted extracellular domain region.

[0157] The present invention provides methods for using DNA 64647encoding LIV-1 polypeptide for the production of compounds inhibitingneoplastic growth as well as for the preparation of screening methodsfor identifying growth inhibitory compounds (e.g. tumor compounds). Inparticular, cDNAs encoding certain LIV-1 polypeptides. For the sake ofsimplicity, in the present specification the proteins encoded by nucleicacid referred to as “DNA 164647”, as well as all further nativehomologues and variants included in the foregoing definition of LIV-1polypeptide, will be referred to as “LIV-1” polypeptide, regardless oftheir origin or mode of expression.

[0158] Cloning of DNA 164647 LIV-1 nucleic acid: Full Length Inverse PCR(“FLIP”), also referred to as inverse long distance PCR because of theability of this method to isolate long genes, was used to clone DNA164647 LIV-1 nucleic acid. The FLIP PCR technique is described inpending U.S. application Ser. No. 09/480,782, filed Jan. 10, 2000. J. C.Grimaldi et al (inventors), Genentech, Inc. (assignee), whichapplication is herein incorporated by reference in its entirety,particularly with respect to the method of cloning a gene. Whilestandard cloning techniques may be used to clone full length genes. FLIPis a very rapid and high-throughput method of isolating an entire clone,vector plus insert, of a specific nucleic acid molecule from any nucleicacid library which was propagated in a host cell that methylates thenucleic acid library. The FLIP cloning method amplifies a target gene ornucleotide sequence and generates a highly purified population of thetarget gene. FIG. 5 is a flow diagram of a FLIP cloning process. DNA164647 was isolated by FLIP methodology using the following primers andprobe. forward primer: (SEQ ID NO:6) LIV1-INV5′5′ ATGTTGACTTGGCAATTTCCACACGGCA 3′ reverse primer: (SEQ ID NO:7)LIV1-INV3′ 5′ TAATGCCAGATTCCCAATTCGGACTAA 3′ probe: (SEQ ID NO:8) LIV1-p5′ TTAGTTCATGAAGGGGATTTGTGACAGAGAGGGCAA AGGTCAGGAT 3′.

[0159] A human LIV-1 gene (Genbank Accession#U41060) has been sequencedby C. Green et al. (direct submission, Nov. 21, 1995). The sequence is3461 bp (SEQ ID NO:1; FIG. 1A) and includes the an open reading frame(ORF). Using the FLIP methodology and the primers and probe disclosedabove, a cDNA clone was isolated from a pool of fifty human cDNAs fromcDNA libraries representing various tissues (Genentech cDNA libraries,Genentech, Inc., So, San Francisco, Calif.). The isolated cDNA cloneincludes a LIV-1 gene comprising a variant nucleic acid sequence (DNA164647; SEQ ID NO:3 (coding sequence) and its complementary sequence)and a variant deduced amino acid sequence (SEQ ID NO:4). The totallength of the isolated LIV-1 DNA 164674 nucleic acid is 2776 bp, thevector pRK5D used was 5.1 kb, thus adding to a total length of 7.9 kb ofthe DNA molecule amplified by IPCR and isolated.

[0160] A nucleic acid sequence comparison of the LIV-1 submitted byGreen et al., supra, and DNA 164647 disclosed herein is shown in FIG. 3.Asterisks denote identity between nucleotides, while dashes representgaps where no identity occurs. An insertion of 18 nucleotides isobserved at approximately nucleotides 446 to 463 of DNA 164647 (SEQ IDNO:3) and two single nucleotide insertions occur at nucleotides 2297 and2323 causing a frame shift and different deduced amino acid sequences inthat region. FIG. 4 is a comparison of the amino acid sequences of theLIV-1 polypeptides of SEQ ID NO:2 (see Green et al., supra) and SEQ IDNO:4 (disclosed herein). A six-amino acid insertion is observed atapproximately amino acids 126 to 131 in the ECD, while different aminoacid sequences are observed at the C-terminal end of the polypeptidesdue to single nucleotide insertions (see amino acids beyond amino acid736 of SEQ ID NO:2 and beyond amino acid 742 of SEQ ID NO:4).

[0161] Specifically; DNA 164647 isolation was performed according to thefollowing procedure. Two adjacent 5′ phosphorylated primers. LIV1-INV5′and LIV1-INV3′, were designed on opposite strands. These primers, SEQ IDNO:6 and SEQ ID NO:7, were used in an inverse PCR reaction. In a 50 ulreaction, the following reagents were added: 50 ng of a bone marrow cDNAlibrary in a modified pRK5D vector, which was propagated in amethylation positive bacteria, 50 picomoles of each PCR primer; 10mmoles of each deoxynucleotide triphosphate, 5 ul of Pfu 10× buffer(Stratagene, La Jolla Calif.), and 1 ul of Pfu Turbo (Stratagene, LaJolla Calif.). The plasmid vector pRK5 (4,661 bp) has been described (EP307,247 published Mar. 15, 1989). The modified pRK5 vector (pRK5.tk.neo)is a derivative of pRK5 in which the neomycin resistance gene isinserted thereby allowing for selection of G418-resistant clones.

[0162] The PCR cycle conditions were one cycle at 94° C. for 3 minutes,then 94° C. for 30 seconds, 65° C. for 30 seconds, 72° C. for 13 minutesfor 20 cycles. PCR conditions may, of course, be modified to meetspecific needs of amplification. The PCR reaction generated a linear 5′phosphorylated amplicon that contained the LIV-1 cDNA insert plus thepRK5D vector.

[0163] Next, 10 ul of the completed PCR reaction was ligated in a 100 ulreaction containing the following other reagents: 10 ul 10×T4 DNA ligasebuffer (New England BioLabs, Beverly, Mass.). 4 ul T4 DNA ligase (NewEngland BioLabs, Beverly, Mass.), 76 ul H₂O. The ligation was allowed toincubate at ambient temperature for 1 hour on the bench top.

[0164] After 1 hour of ligation, 2 ul of the restriction enzyme Dpn1(New England BioLabs, Beverly, Mass.) was added to the ligation reactionand the digestion was allowed to continue for 1 hour at 37° C. Dpn1 willspecifically digest methylated DNA and not unmethylated DNA; therefore,the original cDNA library which was used as a template will be digested,leaving only the LIV-1/vector amplicon intact. After the completion ofthe digestion the sample was cleaned using the QIAquick PCR purificationkit (Qiagen, Valencia, Calif.), eluted in 30 ul of elution buffer orH₂O, and then ethanol precipitated. The pellet was resuspended in 2 ulof H₂O and the entire sample was then used to transformed bacteria.

[0165] Transformation was done by electroporation into DH10B electromaxcompetent bacteria (Life Technologies, Rockville, Md.). The transformedbacteria were plated on Luria broth agar plates and colonies allowed togrow overnight at 37° C.

[0166] The next day, the colonies were lifted onto a nylon membrane,denatured, renatured and probed with a ³²P-ATP kinase-labeled,LIV-1-specific probe (SEQ ID NO:8). DNA from positive colonies wassequenced to confirm that the sequence did not contain point mutationsintroduced by the PCR reaction.

[0167] LIV-1 Polypeptide Production: The description below relatesprimarily to production of LIV-1 polypeptides by culturing cellstransformed or translocated with a vector containing LIV-1-encodingnucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare LIV-1polypeptides. For instance, the LIV-1 polypeptide sequence, or portionsthereof, may be produced by direct peptide synthesis using solid-phasetechniques [see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem.Soc. 85:2149-2154 (1963)]. In vitro protein synthesis may be performedusing manual techniques or by automation. Automated synthesis may beaccomplished, for instance, using an Applied Biosystems PeptideSynthesizer (Foster City, Calif.) using manufacturers instructions.Various portions of the LIV-1 polypeptide may be chemically synthesizedseparately and combined using chemical or enzymatic methods to producethe full-length LIV-1.

[0168] i. Alternative Methods for Synthesis or Isolation ofLIV-1-Encoding DNA

[0169] Disclosed herein are various non-limiting examples of methods forisolating or synthesizing DNA as well as expressing and producingproteins. These methods are applicable and useful for the production andisolation of LIV-1-164647 gene, mRNA, and protein, or fragments thereof.

[0170] DNA encoding LIV-1-164647 polypeptide, homologues, variants, orportions thereof, may be produced by direct DNA synthesis using standardnucleic acid synthetic techniques [see, e.g., Gait, M. J.,Oligonucleotide Synthesis, IRL Press, Oxford, 1984]. DNA synthesis invitro may be performed using manual techniques or by automation.Automated oliogonucleotide synthesis may be accomplished, for instance,using standard techniques. Various portions of the LIV-1-encodingnucleic acid sequence may be chemically synthesized separately andcombined using chemical or enzymatic methods to produce the full-lengthLIV-1-encoding sequence.

[0171] Alternatively, DNA encoding LIV-1 may be obtained from a cDNAlibrary prepared from tissue believed to possess the LIV-1 mRNA and toexpress it at a detectable level. Accordingly, human LIV-1 DNA can beconveniently obtained from a cDNA library prepared from human tissue,such as described in the Examples. The LIV-1-encoding gene may also beobtained from a genomic library or by oligonucleotide synthesis.

[0172] Libraries can be screened with probes (such as antibodies to theLIV-1 polypeptide, or oligonucleotides of at least about 20-80 bases)designed to identify the gene of interest or the protein encoded by it.Screening the cDNA or genomic library with the selected probe may beconducted using standard procedures, such as described in Sambrook etal., Molecular Cloning: A Laboratory Manual (New York: Cold SpringHarbor Laboratory Press, 1989). An alternative means to isolate the geneencoding LIV-1 is to use PCR methodology [Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)].

[0173] The Examples below describe techniques for screening a cDNAlibrary. The oligonucleotide sequences selected as probes should be ofsufficient length and sufficiently unambiguous that false positives areminimized. The oligonucleotide is preferably labeled such that it can bedetected upon hybridization to DNA in the library being screened.Methods of labeling are well known in the art, and include the use ofradiolabels like ³²P-labeled ATP, biotinylation or enzyme labeling.Hybridization conditions, including moderate stringency and highstringency, are provided in Sambrook et al., supra.

[0174] Sequences identified in such library screening methods can becompared and aligned to other known sequences deposited and available inpublic databases such as GenBank or other private sequence databases.Sequence identity (at either the amino acid or nucleotide level) withindefined regions of the molecule or across the full-length sequence canbe determined through sequence alignment using computer softwareprograms such as ALIGN, DNAstar, and INHERIT which employ variousalgorithms to measure homology.

[0175] Nucleic acid having protein coding sequence may be obtained byscreening selected DNA or genomic libraries using the deduced amino acidsequence disclosed herein for the first time, and, it necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

[0176] ii. Selection and Transformation of Host Cells

[0177] Host cells are transfected or transformed with expression orcloning, vectors described herein for LIV-1 production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology; a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

[0178] Methods of transfection are known to the ordinarily skilledartisan, for example, CaPO₄ and electroporation. Depending on the hostcell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes or other cells that contain substantialcell-wall barriers. Infection with Agrobacterium tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23:315 (1983) and WO 89/05859 published Jun. 29, 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 5:456-457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al. J. Bact., 130:946 (1977) and Hsiao et al.,Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods forintroducing DNA into cells, such as by nuclear microinjection,electroporation, bacterial protoplast fusion with intact cells, orpolycations, e.g., polybrene, polyornithine, may also be used. Forvarious techniques for transformine mammalian cells, see Keown et al.,Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature,336:348-352 (1988).

[0179] Suitable host cells for cloning or expressing the DNA in thevectors herein include prokaryote, yeast, or higher eukaryote cells.Suitable prokaryotes include, but are not limited to, eubacteria, suchas Gram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31.537); E. coli strain W3110 (ATCC 27,325) and K5772 (ATCC53,635).

[0180] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forLIV-1-encoding vectors. Saccharomyces cerevisiae is a commonly usedlower eukaryotic host microorganism.

[0181] Suitable host cells for the expression of LIV-1 are derived frommulticellular organisms. Examples of invertebrate cells include insectcells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells.Examples of useful mammalian host cell lines include Chinese hamsterovary (CHO) and COS cells. More specific examples include monkey kidneyCV1 line transformed by SV40 (COS-7. ATCC CRL 1651); human embryonickidney line (293 or 293 cells subcloned for growth in suspensionculture. Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamsterovary cells/−DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)): human lung cells (W138, ATCC CCL 75); human livercells (Hep G2, HB 8065): and mouse mammary tumor (MMT 060562, ATCCCCL51). The selection of the appropriate host cell is deemed to bewithin the skill in the art.

[0182] iii. Selection and Use of a Replicable Vector

[0183] The nucleic acid (e.g., cDNA or genomic DNA) encoding LIV-1 maybe inserted into a replicable vector for cloning (amplification of theDNA) or for expression. Various vectors are publicly available. Thevector may, for example, be in the form of a plasmid, cosmid, viralparticle, or phage. The appropriate nucleic acid sequence may beinserted into the vector by a variety of procedures. In general, DNA isinserted into an appropriate restriction endonuclease site(s) usingtechniques known in the art. Vector components generally include, butare not limited to, one or more of a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence. Construction of suitablevectors containing one or more of these components employs standardligation techniques which are known to the skilled artisan.

[0184] The LIV-1 polypeptide may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavable site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the LIV-1-encoding DNA that is insertedinto the vector. The signal sequence may be a prokaryotic signalsequence selected, for example, from the group of the alkalinephosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published Apr. 4, 1990), or the signal described inWO 90/13646 published Nov. 15, 1990. In mammalian cell expression,mammalian signal sequences may be used to direct secretion of theprotein, such as signal sequences from secreted polypeptides of the sameor related species, as well as viral secretory leaders.

[0185] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells.

[0186] Expression and cloning vectors will typically contain a selectiongene, also termed a selectable marker. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins,e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)complement auxotrophic deficiencies, or (c) supply critical nutrientsnot available from complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

[0187] An example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theLIV-1-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA. 77:4216 (1980). A suitableselection acne for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157(1980)]. The trp1 geneprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

[0188] Expression and cloning vectors usually contain a promoteroperably linked to the LIV-1-encoding nucleic acid sequence to directmRNA synthesis. Promoters recognized by a variety of potential hostcells are well known. Promoters suitable for use with prokaryotic hostsinclude the β-lactamase and lactose promoter systems [Chang et al.,Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,7761, and hybrid promoters suchas the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983)]. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encodingsLIV-1.

[0189] Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem. 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0190] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothioncin, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

[0191] Transcription of LIV-1 from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 publishedJul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-Bvirus and Simian Virus 40 (SV40), from heterologous mammalian promoters,e.g., the actin promoter or an immunoglobulin promoter, and fromheat-shock promoters, provided such promoters are compatible with thehost cell systems.

[0192] Transcription of a DNA encoding a LIV-1 polypeptide by highereukaryotes may be increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp, that act on a promoter to increase its transcription. Manyenhancer sequences are now known from mammalian genes (globin, elastase,albumin, α-fetoprotein, and insulin). Typically, however, one will usean enhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theLIV-1 coding sequence, but is preferably located at a site 5′ from thepromoter.

[0193] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding LIV-1.

[0194] Still other methods, vectors, and host cells suitable foradaptation to the synthesis of LIV-1 in recombinant vertebrate cellculture are described in Gething et al., Nature, 293:620-625 (1981):Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

[0195] iv. Detecting Gene Amplification/Expression

[0196] Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA. 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

[0197] Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal,Conveniently, the antibodies may be prepared against a native sequenceLIV-1 polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to LIV-1DNA and encoding a specific antibody epitope.

[0198] v. Production and Isolation of LIV-1 Polypeptide from Host Cells

[0199] Expression of LIV-1 in Mammalian Cells

[0200] This example illustrates preparation of a LIV-1 polypeptide byrecombinant expression in mammalian cells.

[0201] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), isemployed as the expression vector. Optionally, the LIV-1 DNA 164647 isligated into pRK5 with selected restriction enzymes to allow insertionof the LIV-1 DNA using ligation methods such as described in Sambrook etal., supra. The resulting vector is called pRK5-DNA 164647.

[0202] In one embodiment, the selected host cells may be 293 cells.Human 293 cells (ATCC CCL 1573) are grown to confluence in tissueculture plates in medium such as DMEM supplemented with fetal calf serumand optionally, nutrient components and/or antibiotics. About 10 μgpRK5-DNA 164647 DNA is mixed with about 1 μg DNA encoding the VA RNAgene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μlof 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 1-5 days.

[0203] Approximately 24 hours after the transfections, the culturemedium is removed and replaced with culture medium (alone) or culturemedium containing 200 μCi/ml S-cysteine and 200 μCi/ml ³⁵S-methionine.After a 12 hour incubation, the conditioned medium is collected,concentrated on a spin filter, and loaded onto a 15% (SDS gel. Theprocessed gel may be dried and exposed to film for a selected period oftime to reveal the presence of LIV-1 polypeptide. The culturescontaining transfected cells may undergo further incubation (in serumfree medium) and the medium is tested in selected bioassays.

[0204] In an alternative technique. LIV-1 DNA 164647 may be introducedinto 293 cells transiently using the dextran sulfate method described bySomparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981), 293 cells aregrown to maximal density in a spinner flask and 700 μg pRK5-DNA 164647DNA is added. The cells are first concentrated from the spinner flask bycentrifugation and washed with PBS. The DNA-dextran precipitate isincubated on the cell pellet for four hours. The cells are treated with20% glycerol for 90 seconds, washed with tissue culture medium, andre-introduced into the spinner flask containing tissue culture medium, 5μg/ml bovine insulin and 0.1 μg/ml bovine transferrin, After about fourdays, the conditioned media is centrifuged and filtered to remove cellsand debris. The sample containing expressed LIV-1 can then beconcentrated and purified by any selected method, such as dialysisand/or column chromatography.

[0205] LIV-1 can be expressed in CHO cells. Following PCR amplification,the DNA 164647 is subcloned in a CHO expression vector using standardtechniques as described in Ausubel et al., Current Protocols ofMolecular Biology, Unit 3.16, John Wiley and Sons (1997). CHO expressionvectors are constructed to have compatible restriction sites 5′ and 3′of the DNA of interest to allow the convenient shuttling of cDNA's. Thevector uses expression in CHO cells is as described in Lucas et al.,Nucl. Acids Res. 24: 9 (1774-1779 (1996), and uses the SV40 earlypromoter/enhancer to drive expression of the cDNA of interest anddihydrofolate reductase (DHFR). DHFR expression permits selection forstable maintenance of the plasmid following transfection.

[0206] Approximately twelve micrograms of the desired plasmid DNA isintroduced into approximately 10 million CHO cells using commerciallyavailable transfection reagents, such as Superfect® (Quiagen), Dosper®or Fugene® (Boehringer Mannheim). The cells are grown and described inLucas et al., supra. Approximately 3×10⁻⁷ cells are frozen in an ampulefor further growth and production as described below.

[0207] The ampules containing the plasmid DNA are thawed by placementinto a water bath and mixed by vortexing. The contents are pipetted intoa centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,a 250 mL. 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL.The cell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 was actually used, 3L production spinner is seededat 1.2×10⁶ cells/mL. On day 0, the cell number pH are determined. On day1, the spinner is sampled and sparging with filtered air is commenced.On day 2, the spinner is sampled, the temperature shifted to 33° C. and30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35%polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion).Throughout the production, pH is adjusted as necessary to maintain a pHof about 7.2. After 10 days, or until viability dropped below 70%, thecell culture is harvested by centrifugation and filtering through a 0.22μm filter. The filtrate is either stored at 4° C. or immediately loadedonto columns for purification.

[0208] For the poly-His tagged constructs, the proteins are purifiedusing a Ni-NTA column (Qiagen). Before purification, imidazole is addedto the conditioned media to a concentration of 5 mM. The conditionedmedia is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes,pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rateof 4-5 ml/min. at 4° C. After loading, the column is washed withadditional equilibration buffer and the protein eluted withequilibration buffer containing 0.25 M imidazole. The highly purifiedprotein is subsequently desalted into a storage buffer containing 10 mMHepes. 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine(Pharmacia) column and stored at −80° C.

[0209] LIV-1 may be produced by transient or stable expression in a hostcell, such as COS cells, using standard techniques.

[0210] Expression of LIV-1 in Yeast

[0211] The followings method describes recombinant expression of LIV-1in yeast.

[0212] First, yeast expression vectors are constructed for intracellularproduction or secretion of LIV-1 from the ADH2/GAPDH promoter, DNA164647 encoding a LIV-1 and the promoter is inserted into suitablerestriction enzyme sites in the selected plasmid to direct intracellularexpression of LIV-1. For secretion, DNA encoding LIV-1 can be clonedinto the selected plasmid, together with DNA encoding the ADH2/GAPDHpromoter, a signal peptide, such as a mammalian signal peptide, or, forexample, a yeast alpha-factor or increase secretory signal/leadersequence, and linker sequences (if needed) for expression of LIV-1.

[0213] Yeast cells, such as yeast strain AB110, can then be transformedwith the expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10%, trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

[0214] Recombinant LIV-1 can subsequently be isolated and purified byremoving the yeast cells from the fermentation medium by centrifugationand then concentrating the medium using selected cartridge filters. Theconcentrate containing LIV-1 may further be purified using selectedcolumn chromatography resins.

[0215] Expression of LIV-1 in Baculovirus-Infected Insect Cells

[0216] The following method describes recombinant LIV-1 expression inBaculovirus-infected insect cells.

[0217] The sequence coding for LIV-1 is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-His tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, anucleic acid sequence encoding LIV-1 or the desired portion of thecoding sequence of LIV-1 (such as the sequence encoding theextracellular domain of a transmembrane protein or the sequence encodingthe mature protein if the protein is extracellular) is amplified by PCRwith primers complementary to the 5′ and 3′ regions. The 5′ primer mayincorporate flanking (selected) restriction enzyme sites. The product isthen digested with those selected restriction enzymes and subcloned intothe expression vector.

[0218] Recombinant baculovirus is generated by co transfecting, theabove plasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory ManualOxford: Oxford Universal Press (1994).

[0219] Expressed poly-His tagged LIV-1 can then be purified, forexample, by Ni²⁺-chelate affinity chromatography as follows. Extractsare prepared from recombinant virus-infected Sf9 cells as described byRupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mMMgCl₂; 0.1 mM EDTA; 10% glycerol, 0.1% NP-40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column 0.5 mL per minute. Thecolumn is washed to baseline A₂₈₀ with loading buffer, at which pointfraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH6.0), which elutes non specifically bound portein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or Western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged LIV-1 are pooled and dialyzed againstloading buffer. Alternatively, purification of the IgG tagged (or Fctagged) LIV-1 can be performed using known chromatography techniques,including for instance, Protein A or Protein G column chromatography.

[0220] While the LIV-1 expression is performed in a 0.5-2L scale, it canbe readily scaled up for larger (e.g. 8L) preparations. LIV-1 is alsoexpressed as an IgG construct (immunoadhesin), in which the proteinextracellular region is fused to an IgG1 constant region sequencecontaining the hinge, C_(H)2 and C_(H)3 domains and/or in poly-Histagged forms.

[0221] Following PCR amplification, the coding sequence is subclonedinto a baculovirus expression vector (ph.PH.IgG for IgG fusions andpb.PH.His.c for poly-His tagged proteins), and the vector andBaculogold® baculovirus DNA (Pharmingen) is co-transfected into 105Spondoptera frugiperda (“Sf9”) cells (ATCC CRL 1711), using Lipofectin(Gibco BRL), pb.PH.IgG and pb.PH.His are modifications of thecommercially available baculovirus expression vector pVL1393(Pharmingen), with modified polylinker regions to include the His or Fctag sequences. The cells are grown in Hink's TNM-FH medium supplementedwith 10% FBS (Hyclone). Cells are incubated for 5 days at 28° C. Thesupernatant is harvested and subsequently used for the first viralamplification by injecting Sf9 cells in Hink's TNM-FH mediumsupplemented with 10% FBS at an approximate multiplicity of injection(MOI) of 10. Cells are incubated for 3 days at 28° C. The supernatant isharvested and the expression of the constructs in the baculovirusexpression vector is determined by batch binding of 1 ml, of supernatantto 25 mL of of NI-NTA beads (Qiagen) for histidine tagged proteins orProtein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteinsfollowed by SDS-PAGE analysis comparing to a known concentration ofprotein standard by Coomassie blue staining.

[0222] The first viral amplification supernatant is used to infect aspinner culture (500 mL) of Sf9 cells grown in ESF-921 medium(Expression Systems LLC) at an approximate MOI of 0.1. Cells areincubated for 3 days at 28° C. The supernatant is harvested andfiltered. Batch binding and SDS-PAGE analysis is repeated, as necessary,until expression of the spinner culture is confirmed.

[0223] The conditioned medium from the transfected cells (0.5 to 3 L) isharvested by centrifugation to remove the cells and filtered through0.22 micron filters. For the poly-His tagged constructs, the proteinconstruct are purified using a Ni-NTA column (Qiagen). Beforepurification, imidazole is added to the conditioned media to aconcentration of 5 mM. The conditioned media are pumped onto a 6 mLNi-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3M NaCl and 5 mM imidazole at a flow rate of 4-5 min. at 4° C. Afterloading, the column is washed with additional equilibration buffer andthe protein eluted with equilibration buffer containing 0.25 Mimidazole. The highly purified protein is subsequently desalted into astorage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH6.8, with a 25 mL G25 Superfine (Pharmacia) column and stored at −80° C.

[0224] Immunoadhesin (Fc containing) constructs of proteins are purifiedfrom the conditioned medium as follows. The conditioned medium is pumpedonto a 5 mL Protein A column (Pharmacia) which had been equilibrated in20 mM sodium phosphate buffer, pH 6.8. After loading, the column iswashed extensively with equilibration buffer before elution with 100 mMcitric acid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 mL fractions into tubes containing 275 of 1 M Tris buffer,pH 9. The highly purified protein is subsequently desalted into storagebuffer as described above for the poly-His tagged proteins. Thehomogeneity of the proteins is verified by SDS-PAGE and N-terminal aminoacid sequencing by Edman degradation.

[0225] vi. Purification of Polypeptide

[0226] Forms of LIV-1 polypeptides may be recovered from culture mediumor from host cell lysates. If membrane-bound, it can be released fromthe membrane using a suitable detergent solution (e.g. Triton-X 100) orby enzymatic cleavage. Cells employed in expression of LIV-1 can bedisrupted by various physical or chemical means, such as freeze-thawcycling, sonication, mechanical disruption, or cell lysing agents.

[0227] It may be desired to purify LIV-1 from recombinant cell proteinsor polypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelatine, columns to bind epitope-tagged forms of theLIV-1 polypeptides. Various methods of protein purification may beemployed and such methods are known in the art and described for examplein Deutscher, Methods in Enzymology, 182 (1990); Scopes, ProteinPurification: Principles and Practice, Springer-Verlag, New York (1982).The purification step(s) selected will depend, for example, on thenature of the production process used and the particular LIV-1polypeptide produced.

Example 3 Preparation and Efficacy of Anti-LIV-1 Antibody

[0228] A description follows as to exemplary techniques for theproduction of the anti-LIV-1 antibodies used in accordance with thepresent invention. Techniques for the production of anti-ErbB2antibodies may be found in the ErbB2 publications listed supra andincorporated herein by reference.

[0229] The LIV-1 antigen particularly useful for production ofantibodies may be e.g., a soluble form of the extracellular domain ofLIV-1-164647 or a portion thereof, containing an anitgenic epitope. Theextracellular domain region is indicated in FIG. 1. Alternatively, cellsexpressing LIV-1 at their cell surface can be used to generateantibodies (e.g. NIH-3T3 cells transformed to overexpress LIV-1). Otherforms of LIV-1 useful for generating antibodies will be apparent tothose skilled in the art.

[0230] (i) Polyclonal Antibodies

[0231] Polyclonal antibodies are preferably raised in animals bymultiple subcutaneous (sc) or intraperitoneal (ip) injections of therelevant antigen and an adjuvant. It may be useful to conjugate therelevant antigen to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysing residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

[0232] Animals are immunized against the antigen, immunogenicconjugates, or derivatives by combining, e.g., 100 μg or 5 μg of theprotein or conjugate (for rabbits or mice, respectively) with 3 volumesof Freund's complete adjuvant and injecting the solution intradermallyat multiple sites. One month later the animals are boosted with ⅕ to{fraction (1/10)} the original amount of peptide or conjugate inFreund's complete adjuvant by subcutaneous injection at multiple sites.Seven to 14 days later the animals are bled and the serum is assayed forantibody titer. Animals are boosted until the titer plateaus.Preferably, the animal is boosted with the conjugate of the sameantigen, but conjugated to a different protein and/or through adifferent cross-linking reagent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

[0233] (ii) Monoclonal Antibodies

[0234] Monoclonal antibodies are obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Thus, themodifier “monoclonal” indicates the character of the antibody as notbeing a mixture of discrete antibodies.

[0235] For example, the monoclonal antibodies may be made using thehybridoma method first described by Kohler et al., Nature, 256:495(1975), or may be made by recombinant DNA methods (U.S. Pat. No.4,816,567).

[0236] In the hybridoma method, a mouse or other appropriate hostanimal, such as a hamster, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized is vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp.59-103 [Academic Press, 1986]).

[0237] The hybridoma cells thus prepared are seeded and grown in asuitable culture medium that preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances prevent thegrowth of HGPRT-deficient cells.

[0238] Preferred myeloma cells are those that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred myeloma cell lines are murine myelomalines, such as those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif. USA, and SP-2 or X63-Ag8-653 cells available from the AmericanType Culture Collection, 10801 University Boulevard. Manassas, Va.20110-2209. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodies(Kozbor, J. Immunol., 133:3001 (1984): Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp. 51-63 [MarcelDekker, Inc., New York, 1987]).

[0239] Culture medium in which hybridoma cells are growing is assayedfor production of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

[0240] The binding affinity of the monoclonal antibody can, for example,be determined by the Scatchard analysis of Munson et al., Anal.Biochem., 107:220 (1980).

[0241] After hybridoma cells are identified that produce antibodies ofthe desired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103[Academic Press. 1986]). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

[0242] The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0243] DNA encoding the monoclonal antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5:256-262 (1993) and Plückthun, Immunol. Revs., 130:151-188 (1992).

[0244] In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554(1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 [1992]), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nucl.Acids. Res. 1:2265-2266 [1993]). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

[0245] The DNA also may be modified, for example, by substituting thecoding sequence for human heavy- and light-chain constant domains inplace of the homologous murine sequences (U.S. Pat. No. 4,816,567;Morrison, et al., Proc. Natl Acad. Sci. USA. 81:6851 [1984]), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide.

[0246] Typically such non-immunoglobulin polypeptides are substitutedfor the constant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

[0247] More specifically, an anti-LIV-1 monoclonal antibody is preparedas follows. An anti-LIV-1 IgG₁κ murine monoclonal antibody, preferablyspecific for the extracellular domain of LIV-1-164647 protein, isproduced using procedures like those described in Fendly et al., CancerResearch 5o:1550-1558(1990) and WO89/06692, coupled with ordinary skillin the art. Briefly, LIV-1-expressing cells (preferably cells expressingLIV-1 encoded by DNA 164647) are harvested with phosphate bufferedsaline (PBS) containing 25 mM EDTA and used to immunize BALB/e mice. Themice are given injections i.p. of 10⁷ cells in 0.5 ml PBS on weeks, 0,2, 5 and 7, for example. The mice with antisera that immunoprecipitates³²P-labeled LIV-1 protein, preferably by binding to the extracellulardomain or extracellular fragments, are given i.p. injections of a wheatgerm agglutinin-Sepharose (WGA) purified LIV-1 membrane extract on weeks9 and 13. This is followed by an i.v. injection of 0.1 ml of the LIV-1preparation and the splenocytes are fused with mouse myeloma lineX63-Ag8.653, for example, Hybridoma supernatants are screened forLIV-1-binding by ELISA and radioimmunoprecipitation, MOPC-21 (IgG1),(Cappell, Durham, N.C.), is used as an isotype-matched control. Thedisclosed method of preparing an anti-LIV-1 antibody is provided as anexample since other methods for producing monoclonal antibodies arecontemplated.

[0248] The treatment is performed with a humanized version of the murineanti-LIV-1 antibody. The humanized antibody is engineered by insertingthe complementarity determining regions of the murine anti-LIV-1antibody into the framework of a consensus human immunoglobulin IgG₁(IgG₁) (see for example the process used in Carter et al., Proc. Natl.Acad. Sci. USA 89:4285-4289 [1992]). The resulting humanized anti-LIV-1monoclonal antibody preferably has high affinity for the extracellulardomain of LIV-1 protein and inhibits, in vitro and in vivo and in humanxenografts, the growth of breast cancer cells, lung cancer cells,prostate cancer cells or other cell that overexpresses LIV-1 protein.Preferably the anti-LIV-1 antibodies of the invention inhibit tumor cellgrowth greater than 20%, most preferably greater than 50%, in vitro. Thepreferred anti-LIV-1 monoclonal antibody of the invention is alsoclinically active, as a single agent or in combination with a cytotoxicor other cell growth-inhibiting agent, in patients withLIV-1-overexpressing metastatic breast cancers, or lung, prostate orother cancers. Anti-LIV-1 monoclonal antibody is produced by agenetically engineered Chinese Hamster Ovary (CHO) cell line, grown inlarge scale, that secretes the antibody into the culture medium. Theantibody is purified from the CHO culture media using standardchromatographic and filtration methods. Each lot of antibody is assayedto verity identity, purity, and potency, as well as to meet Food andDrug Administration requirements for sterility and safety.

[0249] When used to kill human cancer cells in vitro for diagnosticpurposes or to test the potency of a lot of antibodies, the antibodieswill typically be added to a culture of LIV-1-overexpressing cells,particularly cancerous cells, that do not also overexpress ErbB2. As acontrol, the antibodies will also be added to a culture of cells that donot overexpress LIV-1. The antibodies are added to the cell culturemedium at a concentration of at least approximately 10 nM. Theformulation and mode of administration for in vitro use are notcritical. Aqueous formulations that are compatible with the culture orperfusion medium will normally be used. Cytotoxicity may be read byconventional techniques to determine the presence or degree of cancer.

[0250] Cytotoxic radiopharmaceuticals for treating cancer may be made byconjugating radioactive isotopes (e.g. I, Y, Pr) to the antibodies. Theterm “cytotoxic moiety” as used herein is intended to include suchisotopes.

[0251] In another embodiment, liposomes are filled with a cytotoxic drugand the liposomes are coated with antibodies specifically binding agrowth factor receptor. Since there are many receptor sites, this methodpermits delivery of large amounts of drug to the correct cell type.

[0252] Antibody dependent cellular cytotoxicity (ADCC) is contemplatedas a method of targeting cytotoxic effects to cancerous cellsoverexpressing LIV-1 protein. The present invention involves a methodbased on the use of antibodies withic are (a) directed against theextracellular domain of LIV-1 protein, and (b) belong to a subclass orisotype that is capable of mediating the lysis of tumor cells to whichthe antibody molecule binds. More specifically, these antibodies shouldbelong to a subclass or isotype that, upon complexing with growth factorreceptors, activates serum complement and/or mediates antibody dependentcellular cytotoxicity (ADCC) by activating effector cells such asnatural killer cells or macrophages.

[0253] The present invention is also directed to the general use ofthese antibodies, in their native form, for therapy of human tumors thatoverexpress the LIV-1 protein. For example, many IgG2a and IgG3 mouseantibodies which bind tumor-associated cell surface antigens can be usedin vivo for tumor therapy. In fact, since LIV-1 is present on a varietyof tumors, the subject antibodies and their therapeutic use have generalapplicability.

[0254] (iii) Humanized and Human Antibodies

[0255] Methods for humanizing non-human antibodies are well known in theart. Preferably, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an import variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986), Riechmnann etal., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 [1988]), by substituting rodent hypervariable regions(e.g., CDRs or CDR sequences for the corresponding sequences of a humanantibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and possibly someFR residues are substituted by residues from analogous sites in rodentantibodies.

[0256] The choice of human variable domains, both light and heavy, to beused in making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol. 196:901[1987]). Another method uses a particular framework region derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA. 89:4285 (1992); Presta et al., J. Immnol. 151:2623 [1993]).

[0257] It is further important that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to a preferredmethod, humanized antibodies are prepared by a process of analysis ofthe parental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

[0258] Alternatively, it is now possible to produce transgenic animals(e.g., mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakohovits et al., Proc. Natl. Acad. Sci. USA. 90:2551(1993); Jakohovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immunol. 7:33 (1993). Human antibodies can also be derivedfrom phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol., 222:581-597 [1991]).

[0259] (iv) Antibody Fragments

[0260] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,Journal of Biochemical and Biophysical Methods 24:107-117(1992) andBrennan et al., Science, 229:81 [1985]). However, these fragments cannow be produced directly by recombinant host cells. For example, theantibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 [1992]). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Other techniques for the production of antibodyfragments will be apparent to the skilled practitioner. In otherembodiments, the antibody of choice is a single chain Fv fragment(scFv). See WO 93/16185.

[0261] (v) Bispecific Antibodies

[0262] Bispecific antibodies are antibodies that have bindingspecificities for at least two different epitopes. Exemplary bispecificantibodies may bind to two different epitopes of the LIV-1 protein. Forexample, one arm may bind a first epitope in the extracellular domain ofLIV-1 protein, while the other may bind a different LIV-1 epitope.Alternatively, an anti-LIV-1 arm may be combined with an arm which bindsto a triggering molecule on a leukocyte such as a T-cell receptormolecule (e.g. CD2 or CD3), or Fc receptors for IgG (FcγR), such asFcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellulardefense mechanisms to the ErbB2-expressing cell. Bispecific antibodiesmay also be used to localize cytotoxic agents to cells which expressLIV-1 by binding to the extracellular domain of the LIV-1 gene product.These antibodies possess a LIV-1 extracellular domain-binding arm and anarm which binds the cytotoxic agent (e.g. saporin, anti-interferon-α,vinca alkaloid, ricin A chain, methotrexate or radioactive isotopehapten). Bispecific antibodies can be prepared as full length antibodiesor antibody fragments (e.g. F(ab′)₂ bispecific antibodies).

[0263] Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 [1983]). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ. 10:3655-3659 (1991).

[0264] According to a different approach, antibody variable domains withthe desired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge. CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immuno-globulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

[0265] In a preferred embodiment of this approach, the bispecificantibodies are composed of a hybrid immunoglobulin heavy chain with afirst binding specificity in one arm, and a hybrid immunoglobulin heavychain-light chain pair (providing a second binding specificity) in theother arm. It was found that this asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one hall of the bispecific molecule provides for afacile way of separation. This approach is disclosed in WO 94/04690. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

[0266] According to another approach described in WO96/27011, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain of an antibody constant domain. In thismethod, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g.tyrosine or tryptophan). Compensatory “cavities” of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers.

[0267] Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

[0268] Techniques for generating bispecific antibodies from antibodyfragments have also been described in the literature. For example,bispecific antibodies can be prepared using chemical linkage. Brennan etal., Science, 229: 81 (1985) describe a procedure wherein intactantibodies are proteolytically cleaved to generate F(ab′)₂ fragments.These fragments are reduced in the presence of the dithiol complexingagent sodium arsenile to stabilize vicinal dithiols and preventintermolecular disulfide formation. The Fab′ fragments generated arethen converted to thionitrobenzoate (TNB) derivatives. One of theFab′-TNB derivatives is then reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for the selectiveimmobilization of enzyme.

[0269] Recent progress has facilitated the direct recovery of Fab′-SHfragments from E. coli, which can be chemically coupled to formbispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992)describe the production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T-cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets. Similar techniques are used to prepare abispecific antibody able to bind cells overexpressing LIV-1 but notoverexpression ErbB2.

[0270] Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA. 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0271] Antibodies with more than two valencies are also contemplated.For example, trispecific antibodies can be prepared. Tutt et al., J.Immunol. 147: 60 (1991).

[0272] (vi) Screening for Antibodies with the Desired Properties

[0273] Techniques for generating antibodies have been described above.Those antibodies having the characteristics described herein areselected.

[0274] To select for antibodies which induce cell death, loss ofmembrane integrity as indicated by, e.g., PI, trypan blue or 7AAD uptakeis assessed relative to control. The preferred assay is the “PI uptakeassay using BT474 cells”. According to this assay, BT474 cells (whichcan be obtained from the American Type Culture Collection [Manassas,Va.]) are cultured in Dulbecco's Modified Eagle Medium (D-MEM):Ham'sF-12 (50:50) supplemented with 10% heat-inactivated FBS (Hyclone) and 2mM L-glutamine. (Thus, the assay is performed in the absence ofcomplement and immune effector cells). The BT474 cells are seeded at adensity of 3×10⁶ per dish in 100×20 mm dishes and allowed to attachovernight. The medium is then removed and replaced with fresh mediumalone or medium containing 10 μg/ml of the appropriate MAb. The cellsare incubated for a 3 day time period. Followings each treatment,monolayers are washed with PBS and detached by trypsinization. Cells arethen centrifuged at 1200 rpm for 5 minutes at 4° C. the pelletresuspended in 3 ml ice cold Ca²⁺ binding buffer (10 mM Hepes, pH 7.4,140 mM NaCl, 2.5 mM CaCl₂) and aliquoted into 35 mm strainer-capped12×75 tubes (1 ml per tube, 3 tubes per treatment group) for removal ofcell clumps. Tubes then receive PI (10 μg/ml). Samples may be analyzedusing a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software(Becton Dickinson). Those antibodies which induce statisticallysignificant levels of cell death as determined by PI uptake areselected.

[0275] In order to select for antibodies which induce apoptosis, an“annexin binding assay using BT474 cells” is available. The BT474 cellsare cultured and seeded in dishes as discussed in the preceding,paragraph. The medium is then removed and replaced with fresh mediumalone or medium containing 10 μg/ml of the MAb. Following a three dayincubation period, monolayers are washed with PBS and detached bytrypsinization. Cells are then centrifuged, resuspended in Ca²⁺ bindingbuffer and aliquoted into tubes as discussed above for the cell deathassay. Tubes then receive labeled annexin (e.g. annexin V-FTIC) (1μg/ml). Samples may be analyzed using a FACSCAN™ flow cytometer andFACSCONVERT™ CellQuest software (Becton Dickinson). Those antibodieswhich induce statistically significant levels of annexin bindingrelative to control are selected as apoptosis-inducing antibodies.

[0276] In addition to the annexin binding assay, a “DNA staining assayusing BT474 cells” is available. In order to perform this assay. BT474cells which have been treated with the antibody of interest as describedin the preceding two paragraphs are incubated with 9 μg/ml HOECHST33342™ for 2 hr at 37° C. then analyzed on an EPICS ELITE™ flowcytometer (Coulter Corporation) using MODFIT LT™ software (VeritySoftware House). Antibodies which induce a change in the percentage ofapoptotic cells which is 2 fold or greater (and preferably 3 fold orgreater) than untreated cells (up to 100% apoptotic cells) may beselected as pro-apoptotic antibodies using this assay.

[0277] To screen for antibodies that bind to an epitope on theextracellular domain of LIV-1-164647, a routine cross-blocking assaysuch as that described in Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed by methods known in theart (see, for example, methods used for the epitope-mapping of theextracellular domain of ErbB2 as determined by truncation mutantanalysis and site-directed mutagenesis (Nakamura et al., J. of Virology67(10):6179-6191 [October 1993]; Renz et al., J. Cell Biol.125(6):1395-1406 [June 1994]). In addition, where a particular aminoacid sequence is suspected of forming all or a substantial portion of anepitope, a polypeptide consisting of that sequence may be contacted withthe antibody and tested, using standard techniques, for its ability tocompete for binding to the LIV-1-164647, such as the LIV-1-164647 ECD.

[0278] To identify anti-LIV-1 antibodies which inhibit growth ofLIV-1-expressing cells in cell culture by 50-100%, an assay can beperformed generally as follows: LIV-1-expressing cells are grown in a1:1 mixture of F12 and DMEM medium supplemented with 10% fetal bovineserum, glutamine and penicillinstreptomycin. The LIV-1-expressing cellsare plated at 20,000 cells in a 35 mm cell culture dish (2 mls/35 mmdish), 2.5 μg/ml of the anti-LIV-1 antibody is added per dish. After sixdays, the number of cells, compared to untreated cells are counted usingan electronic COULTER™ cell counter. Those antibodies which inhibitgrowth of the LIV-1-expressing cells by 50-100% are selected forcombination with the apoptotic antibodies as desired.

[0279] (vii) Effector-Function Engineering

[0280] It may be desirable to modify the antibody of the invention withrespect to effector function, so as to enhance the effectiveness of theantibody in treating cancer, for example. For example, cysteineresidue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated may have improved internalization capabilityand/or increased complement-mediated cell killing and antibody-dependentcellular cytotoxicity (ADCC). See Caron et al., J. Exp Med.176:1191-1195 (1992) and Shopes. B. J. Immunol. 148:2918-2922 (1992).Homodimeric antibodies with enhanced anti-tumor activity may also beprepared using heterobifunctional cross-linkers as described in Wolff etal., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody canbe engineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design 3:219-230 (1989).

[0281] (viii) Immunoconjugates

[0282] The invention also pertains to immunoconjugates comprising theantibody described herein conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g. an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

[0283] Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated anti-ErbB2 antibodies. Examples include ²¹²Bi, ¹³¹I,¹³¹In, ⁹⁰Y and ¹⁸⁶Re.

[0284] Conjugates of the antibody and cytotoxic agent are made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelatine ardent forconjugation of radionucleotide to the antibody. See WO94/11026.

[0285] In another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g. avidin) whichis conjugated to a cytotoxic agent (e.g. a radionucleotide).

[0286] (ix) Immunoliposomes

[0287] The anti-LIV-1 antibodies disclosed herein may also be formulatedas immunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA. 82:3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77:4030 (1980): and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

[0288] Particularly useful liposomes can be generated by the reversephase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257: 286-288 (1982) via a disulfide interchange reaction Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al., J. National Cancer Inst. 81(19)1484 (1989).

[0289] (x) Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

[0290] The antibodies of the present invention may also be used in ADEPTby conjugating the antibody to a prodrug-activating enzyme whichconverts a prodrug (e.g. a peptidyl chemotherapeutic agent, seeWO81/01145) to an active anti-cancer drug. See, for example, WO 88/07378and U.S. Pat. No. 4,975,278.

[0291] The enzyme component of the immunoconjugate useful for ADEPTincludes any enzyme capable of acting on a prodrug in such a way so asto covert it into its more active, cytotoxic form.

[0292] Enzymes that are useful in the method of this invention include,but are not limited to alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into tree drugs; cytosingdeaminase useful for converting non-toxic 5-fluorocytosing into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxy peptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuramimidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs, Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature 328:457-458 [1987]). Antibody-abzyme conjugates can be prepared as describedherein for delivery of the abzyme to a tumor cell population.

[0293] The enzymes of this invention can be covalently bound to theanti-LIV-1 antibodies by techniques well known in the art such as theuse of the heterobifunctional crosslinking reagents discussed above.Alternatively, fusion proteins comprising at least the antigen bindingregion of an antibody of the invention linked to at least a functionallyactive portion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 312: 604-608 [1984]).

[0294] (xi) Antibody-Salvage Receptor Binding Epitope Fusions

[0295] In certain embodiments of the invention, it may be desirable touse an antibody fragment, rather than an intact antibody, to increasetumor penetration, for example. In this case, it may be desirable tomodify the antibody fragment in order to increase its serum hall life.This may be achieved, for example, by incorporation of a salvagereceptor binding epitope into the antibody fragment (e.g. by mutation ofthe appropriate region in the antibody fragment or by incorporating theepitope into a peptide tag that is then fused to the antibody fragmentat either end or in the middle, e.g., by DNA or peptide synthesis).

[0296] A systematic method for preparing such an antibody variant havingan increased in vivo half-life comprises several steps. The firstinvolves identifying the sequence and conformation of a salvage receptorbinding epitope of an Fc region of an IgG molecule. Once this epitope isidentified, the sequence of the antibody of interest is modified toinclude the sequence and conformation of the identified binding epitope.After the sequence is mutated, the antibody variant is tested to see ifit has a longer in vivo half-life than that of the original antibody. Itthe antibody variant does not have a longer in vivo half-life upontesting, its sequence is further altered to include the sequence andconformation of the identified binding epitope. The altered antibody istested for longer in vivo half-life, and this process is continued untila molecule is obtained that exhibits a longer in vivo half-life.

[0297] The salvage receptor binding epitope being thus incorporated intothe antibody of interest is any suitable such epitope as defined above,and its nature will depend, e.g., on the type of antibody beingmodified. The transfer is made such that the antibody of interest stillpossesses the biological activities described herein.

[0298] The epitope preferably constitutes a region wherein any one ormore amino acid residues from one or two loops of a Fc domain aretransferred to an analogous position of the antibody fragment. Even morepreferably, three or more residues from one or two loops of the Fcdomain are transferred. Still more preferred, the epitope is taken fromthe C_(H)2 domain of the Fc region (e.g., of an IgG) and transferred tothe C_(H)1, C_(H)3, or V_(H) region, or more than one such region, ofthe antibody. Alternatively, the epitope is taken from the C_(H)2 domainof the Fc region and transferred to the C_(L) region or V_(L) region, orboth, of the antibody fragment.

[0299] (xii) Purification of Anti-LIV-1 Antibody

[0300] When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. It the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration, Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5). EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems arepreferably first concentrated using a commercially available proteinconcentration filter, for example an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

[0301] The antibody composition prepared from the cells can be purifiedusing, for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purity antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 [1983]). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 [1986]). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation. Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

[0302] Following any preliminary purification step(s), the mixturecomprising the antibody of interest and contaminants may be subjected tolow pH hydrophobic interaction chromatography using an elution buffer ata pH between about 2.5-4.5, preferably performed at low saltconcentrations (e.g. from about 0-0.25M salt).

Example 4 LIV-1 is Expressed on the Cell Surface

[0303] Analysis of the deduced amino acid sequence of LIV-1 indicatedthat a portion of the protein exists as an extracellular domain (ECD).To verify this finding, the ECD was expressed, purified, and used as anantigen for the development of anti-LIV-1 ECD antibodies. The antibodieswere contacted with cells expressing the full length LIV-1 protein. ThisExample shows that anti-LIV-1 ECD antibodies bound to LIV-1-expressingcells, demonstrating that LIV-1 comprises an extracellular domain.

[0304] Expression of LIV-1 ECD in E. coli

[0305] To obtain samples of the LIV-1 ECD to which antibodies could beraised, a polypeptide comprising the extracellular domain of LIV-1 wasexpressed in E. coli after first constructing a nucleic acid vectorencoding the LIV-1 extracellular domain operably linked to an amino acidleader sequence. The following procedures were used to prepare thenucleic acid construct and express the encoded protein.

[0306] DNA coding for amino acids 1-298, encoding the extracellulardomain of mature LIV-1, was prepared by standard PCR techniques from afull length cDNA clone using the primers5′-CAACATCAAATGCATCAACTTCATGAACTAAAAGCAGCTGCT-3′ (SEQ ID NO:9) and5′-GAGCTCGAGCGGCCGCTTAGGTCTTTGGAGGGATTTCAGCCTT-3′ (SEQ ID NO:10). ThePCR reaction was divided in two: one half was digested with NsiI andSacI, while the other half was digested with SacI and NotI. TheNsiI-SacI DNA fragment encoding amino acids 1-166 and the Such-NotIfragment encoding amino acids 167-298 were isolated and ligated into thepreviously digested expression vector pST239, a pBR322-derived vectorcontaining an N-terminal polyhis leader at the 3′ end of which is anNsiI restriction site. The resulting LIV-1 ECD expression plasmid wasdesignated pE164647.

[0307] Transcriptional and translational control of expression in theST239 vector were provided by the following genetic features.Transcriptional initiation was controlled by the E. coli alkalinephosphatase promoter (Kikuchi Y. at al., Nucleic Acids Res. 9:5671-5678(1981)). The trp operon ribosome binding site was used for translationinitiation (Yanofsky C. et al., Nucleic Acids Res. 9:6647-6668 (1981)).Translational termination was effected by the translation terminationcodon and the downstream λto transcriptional terminator (Scholtissek S.et al., Nucleic Acids Res. 15:3185 (1987)), followed by the rare codontRNA genes pro2, argU, and glyT (Komine Y., et al., J. Mol. Biol.212:579-598(1990), Fournier M. J., et al. Microbiol. Rev. 49:379-397(1985)).

[0308] To facilitate expression, the extracellular domain of LIV-1 wasexpressed in E. coli cytoplasm with a N-terminal poly-histidine leadersequence encoded within the ST239 expression vector. The amino acidsequence of this leader was: MK HQHQHQHQHQHIQMHQ (SEQ ID NO:111) Thisleader sequence offered several advantages. First, translationinitiation was optimized. In addition, purification was simplified byadsorption on a nickel chelation column. Finally, the leader sequencewas easily and efficiently removed, as desired, using the TAGZyme™system (Unizyme Laboratories, Horsholn, Denmark).

[0309] Following construction of the expression plasmid, pE164647, andDNA sequence verification, the LIV-1 expression plasmid was transformedinto the E. coli strain 58F3 (fhuAΔ(tonAΔ)tonΔ galE rpoHts(hipRts)ΔclpPlacIq ΔompTΔ(nmpc-fepE)ΔslyD). Transformants were initially cultured inLuria broth at 30° C. overnight, and then diluted 100-fold into aphosphate-limiting media to induce the alkaline phosphatase promoter.After 24 hours at 30° C. with shaking, the cultures were centrifuged,and the cell pastes frozen until the start of purification.

[0310] Purification of LIV-1 ECD

[0311] A 0.5 liter fermentation of E. coli transformants expressingLIV-1 yielded approximately 6-10 grams of cell paste. The paste wasresuspended in 10 volumes (w/v) of 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate were added to makefinal concentrations of 0.1 M and 0.02 M. respectively, and the solutionwas stirred overnight at 4° C. This sulfitolysis step resulted in adenatured protein having all cysteine residues blocked. The solution wascentrifuged at 40 K rpm in a Beckman ultracentifuge for 30 min. Thesupernatant was diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris. pH 7.4) and filtered through 0.22 micronfilters to clarify. A volume corresponding to 50 mls of the clarifiedextract was loaded onto a 5 ml Qiagen Ni-NTA (nickel-nitrilotriaceticacid) metal chelate affinity column equilibrated in the metal chelatecolumn buffer (QIAexpress® Protein Purification System, Qiagen,Valencia, Calif. USA). The column was washed with additional buffercontaining 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. Theprotein was eluted with buffer containing 250 mM imidazole. Fractionscontaining the desired protein were pooled and stored at 4° C. Proteinconcentration was estimated by its absorbance at 280 nm using thecalculated extinction coefficient based on its amino acid sequence.Protein for antibody production was obtained by reducing an aliquot ofthe Ni-NTA pool with dithiothreitol (50 mM final concentration) followedby extensive dialysis against 1 mM HCl.

[0312] Development of Monoclonal Antibodies to LIV-1 Protein

[0313] Monoclonal antibodies specific for the extracellular domain ofLIV-1 were developed according to the following procedures.

[0314] Ten Balb/c mice (Charles River Laboratories, Wilmington, Del.)were hyperimmunized with recombinant polyhistidine-tagged human LIV-1isolated from pE164647-transformed E. coli as described herein. Thetagged LIV-1 protein in Ribi adjuvant (Ribi Immunochem Research, Inc.,Hamilton, Mo.) was administered to the mice. B-cells from five micedemonstrating high anti-LIV-1 antibody titers were fused with mousemyeloma cells (X63.Ag8.653; American Type Culture Collection, Rockville,Md.) using a modified protocol analogous to one previously described(Kohler. G. and Milstein, C., Nature 256: 495-497 (1975): Hongo, J. S.et al., Hybridoma 14:253-260 (1995)).

[0315] After 7-14 days, the supernatants were harvested and screened forantibody production and LIV-1 binding specificity by standard techniquesusing direct enzyme-linked immunosorbent assay (ELISA). Sixteen positiveclones, showing the highest immunobinding affinity after the secondround of subcloning by limiting dilution, were injected intoPristane-primed mice (Freund, Y. R. and Blair, P. B., J. Immunol.129:2826-2830(1982)) for in vivo production of monoclonal antibody. Theascites fluids from these mice were pooled and purified by Protein Aaffinity chromatography (Pharmacia fast protein liquid chromatography(FPLC) Pharmacia, Uppsala, Sweden) as previously described (Hongo, J. S.et al., Hybridoma, supra (1995)). The purified antibody preparationswere sterile filtered (0.2-μm pore size; Nalgene, Rochester N.Y.) andstored at 4° C. in phosphate buffered saline (PBS).

[0316] Expression of LIV-1 ECD Fragments in E. coli

[0317] Fragments of the LIV-1-164647 ECD were expressed in E. coli usingthe same techniques as described for cloning and expressing the fulllength LIV-1-164647. The expressed ECD fragments were the N-terminalfragment (amino acid 1 to and including amino acid 147 of SEQ ID NO:4)and the C-terminal fragment (amino acid 148 to and including amino acid298 of SEQ ID NO:4). The fragments were purified as described above.

[0318] Standard Western blot analysis was used to determine which of theN-terminal or C-terminal ECD fragments bound to monoclonal antibodiesisolated as described above. Only monoclonal antibodies 2945, 2982,2984, 2985, 2987, and 2988 were tested. It was found that monoclonalantibody 2984 bound to the N terminal fragment, while monoclonalantibody 2945, 2982, 2985, 2987, and 2988 bound to the C-terminalfragment. The results are listed in Table 3.

[0319] Expression of Exogenous Full-Length LIV-1 in Mammalian Cells

[0320] Each of the 16 antibodies listed in Table 3 was examined forbinding to the ECD of LIV-1-164647 and found to bind specifically. Theantibodies were characterized to have the properties listed in Table 3.Epitope characterization involved determination as to whether a testantibody can compete for binding to the same epitope as the epitopebound by an anti-LIV-1-164647 antibody of the present invention,including the antibodies produced by the hybridomas deposited with theATCC, using a cross-blocking (e.g., a competitive ELISA assay) can beperformed. In an exemplary competitive ELISA assay, LIV-1-167647 or itsECD (or other fragment) coated on the wells of a microtiter plate ispre-incubated with or without candidate competing antibody and then thebiotin-labeled anti-LIV-1-164647 antibody of the invention is added. Theamount of labeled anti-LIV-1-164647 antibody bound to the LIV-1 antigenin the wells is measured using avidin-peroxidase conjugate andappropriate substrate. The antibody can be labeled with a radioactive orfluorescent label or some other detectable and measurable label. Theamount of labeled anti-LIV-1 antibody bound to the antigen has anindirect correlation to the ability of the candidate competing antibody(test antibody) to compete for binding to the same epitope (i.e., thegreater the affinity of the test antibody for the same epitope, the lessof the labeled antibody will be bound to the antigen-coated wells). Acandidate competing antibody is considered an antibody that bindssubstantially to the same epitope or that competes for binding to thesame epitope as an anti-LIV-1 antibody of the invention if the candidateantibody can block binding of the LIV-1 antibody by at least 20%,preferably by at least 20-50%, even more preferably, by at least 50% ascompared to the control performed in parallel in the absence of thecandidate competing antibody (but may be in the presence of a knownnon-competing antibody). It will be understood that variations of thisassay can be performed to arrive at the same quantitative value. Theepitope group assignment for each antibody in Table 3 was determined bycompetitive ELISA. Each antibody was biotinylated and tested for bindingto LIV-1-164647 ECD coated on plates in the presence or absence of anexcess of each unlabeled anti-LIV-1-164647 monoclonal antibody.Streptavidin-HRP was then added to the plates followed by peroxidasesubstrate. A decrease in the binding by at least 50% or a lack ofbinding of biotinylated monoclonal antibodies to LIV-1-164647 ECDindicated that both unlabeled and biotinylated antibodies bound to thesame (or proximal) epitope on LIV-1-164647.

[0321] Expression of full-length LIV-1 in mammalian cells resulted inthe exposure of the extracellular domain on the surface of the cells. Todemonstrate this, 3T3 cells were transiently transfected with anexpression construct encoding wild-type full length LIV-1646471(pRK5-LIV-1-164647). Control cells were transfected with the pRK5 vectorlacking the LIV-1 insertion. After 24 hours, cells were analyzed forcell surface expression by fluorescent activated cell sorting (FACS)analysis using the anti-LIV-1-164647 ECD monoclonal antibody, describedherein, as a tag. Approximately 10⁶ cells were incubated for 30 min onice in PBS containing 2% Goat serum and 5% Rabbit serum (FACS buffer)then for 2 hours in FACS buffer containing 1 μg/ml of anti-LIV-1-164647ECD monoclonal antibody 2982 (isolated from hybridoma 2982.4A12.1E8.1C4, designated ATCC ______) or anti-LIV-1-164647 ECD monoclonalantibody 2983 (isolated from hybridoma 2983.3G9.1D4.1D7.ATCC ______).The cells were then washed with ice cold PBS, incubated for 20 minutesat 4° C. with a Biotin-conjugated Goat anti-human IgG second antibody(Jackson Immunoreagents: West Grove, Pa.), washed before incubation for20 min at 4° C. with phycoerythrin conjugated streptavidin (JacksonImmunoreagents, West Grove, Pa.). Cells were washed again prior tocytofluorometry. The FACS analysis results using antibody 2983 areplotted in FIG. 6. The results demonstrated that cell surface expressionof LIV-1-164647 was detected in 3T3 cells transfected withpRK5-LIV-1-164647, but not in control (pRK5 vector only) cells. Thus,amino acids 1-298 of the LIV-1-164647 protein constitute anextracellular domain.

[0322] Expression of Endogenous LIV-1 in a Breast Tumor Cell Line

[0323] It is disclosed herein that endogenous LIV-1 is expressed on thesurface of cells of the MCF-7 breast tumor cell line (ATCC HTB-22, forexample). Anti-LIV-1 monoclonal antibody 2945 (from hybridoma2945.2G1.1C7.2F10: ATCC ______) was radiolabeled with ¹²⁵I using thelactoperoxidase method. MCF-7 cells were harvested with PBS containingmM EDTA. A population of 3.4 million MCF-7 cells were incubated in PBSA(PBS+0.1% BSA+0.02% azide) with 50 000 cpm (230 pM) of ¹²⁵I-2945 in thepresence (NSB) or absence (Tot) of an excess of unlabeled antibody (0.25μM) for 1 h at room temperature. Bound radioactivity was then separatedfrom unbound radioactivity by centrifugation for 5 min at 5000 rpm overa 1 ml cushion of 20% sucrose in cold PBS. Radioactivity associated withthe cell pellet was then counted in a gamma counter. As shown in FIG. 7,specific binding of anti-LIV-1-164647 monoclonal antibody 2945 wasdetected at the surface of MCF-7 cells indicating that endogenous LIV-1is expressed at the cell surface in this breast tumor cell line.

[0324] Antibody Binding to LIV-1

[0325] Selected monoclonal antibodies of the present invention and theirrespective hybridomas are listed in Table 3. Characterization of themonoclonal antibodies was performed as described herein using standardtechniques of competition binding for epitope analysis, cell sorting forFACS analysis, and Western blotting for determining whether the antibodyhound to the N-terminal or C-terminal portion of the LIV-1-164647 ECD.TABLE 3 L1V-1-Binding Monoclonal Antibodies Monoclonal Hybridoma EpitopeFACS ECD fragment Antibody Cell Line Isotype Group^(a) (3T3-LIV-1/3T3)N- or C-terminal^(b) 2945 2G1.1C7.2F10 IgG2b A +++/− C-terminal 29824A12.1E8.1C4 IgG1 A +++/− C-terminal 2983 3G9.1D4.1D7 IgG1 A +++/− nottested 2984 6D6.1H10.2C1 IgG1 C +++/− N-terminal 2985 4F3.2D6.1D7 IgG1 A+/− C-terminal 2987 1D8.1C11.2B7 IgG1 C ++/− C-terminal 2988 1A7.1F2.1H7IgG1 A +/− C-terminal 2946 2B11.2B11.2A12 IgG2b B −/− not tested 29473H8.2D9.1H8 IgG1 B ++/− ″ 2948 2G4.2C7.2D6 IgG2b B −/− ″ 29495B4.2H11.1G10 lgG2b B −/− ″ 2950 5H5.2A7.1D8 IgG2b B −/− ″ 29514G3.2F8.2A11 IgG2b B ++/− ″ 2952 6G9.1G9.1A10 leG2b B −/− ″ 29536B6.2E11.1F10 IgG1 B +/− ″ 2986 1F1.2G8.2E7 IgG1 B −/− ″

[0326] Deposit of some of these hybridomas with the American TypeCulture Collection under the Budapest Treaty is described herein underthe heading “Deposit of Material.”

Example 5 LIV-1 Expression in Tumor Tissue Examined by RNA in SituHybridization

[0327] This example provides methods used in the preparation of tissuearrays for the determination of LIV-1 expression in various humantissues (see, for example, Kononen, J., et al. Nature Medicine 4:844-847(1998)).

[0328] Preparation of Tissue Microarrays

[0329] A tissue microarray, or tissue array, is a paraffin blockcontaining several individual tissue samples. A typical tissuemicroarray may contain 1000 or more samples. Tissue microarrays allowthe examination of a large series of specimens while maximizingefficient utilization of technician time, reagents, and valuable tissueresources.

[0330] Tissue microarrays are constructed by first removing small cores(0.6 mm diameter. 3-4 mm height) from “donor” tissue biopsy samplesembedded in paraffin blocks using a tissue array instrument (BeecherInstruments, Silver Spring, Md., USA). Using the same instrument, eachcore sample is then re-embedded, together with other biopsy cores, in asingle “recipient” block to form an array. Preferably, each tissue issampled in triplicate. Thin slices (4-8 μm thick) of a recipient blockwere mounted on glass slides. Visualization and screening, may beperformed by histological methods including, but not limited to,standard hematoxylin and cosin staining for morphological analysis;immunohistochemitry (IHC) for protein detection; mRNA in situhybridization (mRNA ISH) and RT-PCR for mRNA detection; fluorescence insitu hybridization (FISH) and in situ PCR for DNA detection; andterminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-endlabeling (TUNEL) assay for detection of cells undergoing apoptotic DNAfragmentation.

[0331] The followings mRNA ISH procedures were used to determine a humantissue expression profile for LIV-1.

[0332] In Situ Hybridization

[0333] In situ hybridization is a powerful and versatile technique forthe detection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identity sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis and aid in chromosome mapping.

[0334] In situ hybridization was performed following an optimizedversion of the protocol by Lu and Gillett, Cell Vision 1: 169-176(1994), using PCR-generated ³³ P-labeled riboprobes. Briefly,formalin-fixed, paraffin-embedded human tissues were sectioned,deparaffinized, deproteinated in proteinase K (20 μg/ml) for 15 minutesat 37° C., and further processed for in situ hybridization as describedby Lu and Gillett, supra. A [³³-P] UTP-labeled antisense riboprobe wasgenerated from a PCR product and hybridized at 55° C. overnight. Theslides were dipped in Kodak NTB2 nuclear track emulsion and exposed for4 weeks.

[0335] Preparation of DNA Template

[0336] For synthesis of a LIV-1-specific riboprobe, template DNAcorresponding to a portion of LIV-1 was needed. To that end, a portionof the DNA 164647 (LIV-1-164647) nucleic acid sequence from nucleotide1690-nucleotide 2240 (SEQ ID NO:3; see FIG. 2A) having the followingsequence was amplified for use as template DNA: 5′-TGCCATTCAC ATTTCCACGATACACTCGGC CAGTCAGACG ATCTCATTCA CCACCATCAT GACTACCATC ATATTCTCCATCATCACCAC CACCAAAACC ACCATCCTCA CAGTCACAGC CAGCGCTACT CTCGGGAGGAGCTGAAAGAT GCCGGCGTCG CCACTTTGGC CTGGATGGTG ATAATGGGTG ATGGCCTGCACAATTTCAGC GATGGCCTAG CAATTGGTGC TGCTTTTACT GAAGGCTTAT CAAGTGGTTTAAGTACTTCT GTTGCTGTGT TCTGTCATGA GTTGCCTCAT GAATTAGGTG ACTTTGCTGTTCTACTAAAG GCTGACATGA CCGTTAAGCA GGCTGTCCTT TATAATGCAT TGTCAGCCATGCTGGCGTAT CTTGGAATGG CAACAGGAAT TTTCATTGGT CATTATGCTG AAAATGTTTCTATGTGGATA TTTGCACTTA CTGCTGGCTT ATTCATGTAT GTTGCTCTGG TTGATATGGTACCTGAAATG CTGCACAATG ATGCTAGTGA CCATGGATGT AGCCGCTGGG G-3′ (SEQ IDNO:12). The amplified DNA was used as the template from which aradiolabeled riboprobe was synthesized for in situ hybridization.

[0337] Primers used for generating an antisense cDNA strand of LIV-1from template nt 1690 nt 2240:

[0338] Primer F-99104: 5′-GGA TTC TAA TAC GAC TCA CTA TAG GGC TGC CATTCA CAT TTC CAC GAT-3′ (SEQ ID NO:13).

[0339] Primer F-99105: 5′-CTA TGA AAT TAA CCC TCA CTA AAG GGA CCC CAGCGC CTA CAT CC-3′ (SEQ ID NO:14).

[0340] The Advantage cDNA polymerase mix from Clonetech (8417-1) wasused according to the manufacturer s directions with slightmodifications. Briefly, 316 μl SQ water (highly purified, RNase-freewater), 40 μl 10×PCR buffer, 16 μl 10 mM dNT), 8 μl primer SEQ ID NO:13,8 μl primer SEQ ID NO:14 were combined to form a master mixture. Fromthe master mixture, 97 μl were aliquoted into a PCR tube followed by theaddition of 2 μl of template DNA and 1 μl of Advantage cDNA polymerase.Using a Perkin-Elmer Cetus 9600 thermocycler, cycle conditions were asfollows:

[0341] Begin: 85° C., 5 minutes

[0342] 60° C., 1.5 minutes

[0343] 10 cycles of:

[0344] 94° C. 30 seconds

[0345] 68° C. 30 seconds

[0346] 72° C. 1 minutes

[0347] 15 cycles of:

[0348] 94° C. 30 seconds

[0349] 55° C. 30 seconds

[0350] 72° C. 1 minutes

[0351] followed by:

[0352] 72° C. 7 minutes

[0353] 4° C. hold

[0354] Upon completion of the PCR cycles, the PCR product was filterthrough a Microcon-50™ filter unit to remove primers and excess buffer.

[0355] LIV-1 ³³P-Riboprobe Synthesis

[0356] 6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol)were speed vac dried. To each tube containing dried ³³P-UTP, thefollowing ingredients were added:

[0357] 2.0 μl 5× transcription buffer

[0358] 10 μl DTT (100 mM)

[0359] 2.0 μl NTP mix (2.5 mM; 10 μl; each of 10 mM GTP, CTP & ATP+10 μlH₂O)

[0360] 1.0 μl Rnasin ribonuclease inhibitor

[0361] 3.0 μl DNA template (1 μg) in H₂O

[0362] 1.0 μl RNA polymerase (for PCR products T3=AS (antisense), T7=S(sense), usually)

[0363] The tubes were incubated at 37° C. for one hour, 1.0 μl RQ1 DNasewas added followed by incubation at 37° C. for 15 minutes, 90 μl TE (10mM Tris pH 7.6/1 mM EDTA pH 8.0) were added, and a 1.0 μl aliquot of themixture was pipetted onto DE81 paper. The remaining solution was loadedin a Microcon-50 ultrafiltration unit (Amicon, 42416), and spun 6minutes using program 10 of a Heraeus Centrifuge 28RS. The filtrationunit was inverted over a second tube and spun for 3 minutes usingprogram 2. After the final recovery spin, 100 μl TE were added, 1 μl ofthe final product was pipetted on DE81 paper. The samples dotted ontoDE81 paper before and after filtration were counted in 6 ml of BiofluorII in a Beckman LS 5000TD scintillation counter.

[0364] To verify its size, the probe was run on a TBE/urea gel. 1-3 μlof the filtered probe or 5 μl of RNA Molecular Weight Marker III(Boehringer Mannheim) were added to 3 μl of loading butter. Afterheating on a 37° C. heat block for three minutes, the probes wereimmediately placed on ice. The wells of gel were flushed, the samplesloaded, and run at 180-250 volts for 45 minutes. The gel was wrapped insaran wrap and exposed to Biomax MS™ film or XAR-2™ film (Kodak) with anintensifying screen in −70° C. freezer one hour to overnight. The LIV-1riboprobe thus prepared was designated 764 AS or 764 S for the antisenseand sense probes, respectively.

[0365]³³P-Hybridization

[0366] Pretreatment of paraffin-embedded sections: The thin slices of arecipient tissue array block mounted on glass slides weredeparaffinized, placed in SQ H₂O, and rinsed twice in 2×SSC at roomtemperature, for 5 minutes each time. The sections were deproteinated in20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 ml RNase-free RNasebuffer) at 37° C. for 15 minutes). Slides were subsequently rinsed in0.5×SSC, dehydrated through graded ethanols (70%, 95%, and 100%) for 2minutes at each grade, and air-dryed.

[0367] Prehybridization: The slides were laid out in plastic box linedwith Box buffer (4×SSC, 50% formamide)—saturated filter paper. Thetissue was covered with 100 μl of hybridization buffer (10% (DextranSulfate, 50% formamide, 1×SSC) and incubated at 42° C. for 1-4 hours.

[0368] Hybridization: 2.0×10⁶ cpm probe and 2.0 μl tRNA (100 mg/mlstock) per slide were heated at 95° C. for 3 minutes. The slides werecooled on ice, and hybridization buffer was added to make a final volumeof 100 μl per slide. After vortexing, 100 μl ³³P mix were added to 100μl prehybridization on slide. The slides were incubated overnight at 55°C.

[0369] Washes: Washing was done 2×10 minutes with 2×SSC, EDTA at roomtemperature (400 ml 20×SSC+16 ml 0.25M EDTA, V_(f)=4L), followed byRnase treatment at 37° C. for 30 minutes (500 μl of 10 mg/ml in 250 mlprewarmed Rnase buffer=20 μg/ml). The slides were washed 2×10 minuteswith 2×SSC, EDTA at room temperature. The stringency wash conditionswere as follows: 4×30 minutes at 55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16ml EDTA, V_(f)=4L). This was followed by 2×10 minute washes 0.5SSc atRT. The slides were then dehydrated for 2 minutes at each of 50%, 70%,90% ethanol contain 0.3 M ammonium acetate, and air dried for 2 hours.The dried slides were exposed to Biomax MS film (Kodak) for 16 hours orHyperfilm β-Max film (Amersham) over 2 days.

[0370] ErbB2 and β-actin ³³P-Riboprobe Synthesis

[0371] For comparative analysis of LIV-1 and ErbB2 expression by RNA insitu hybridization, a riboprobe complementary to an ErbB2 nucleic acidsequence was prepared. As a control, expression of β-actin was alsomonitored using a riboprobe complementary to the RNA of that gene.

[0372] Using the procedures just disclosed, riboprobes specific forErbB2 and β-actin were also prepared. The ErbB2 riboprobe wassynthesized by transcription from a DNA template having the followingsequence, 5′-TGGTCGTGGT CTTGGGGGTG GTCTTTGGGA TCCTCATCAA GCGACGGCAGCAGAAGATCC GGAAGTACAC GATGCGGAGA CTGCTGCAGG AAACGGAGCT GGTGGAGCCGCTGACACCTA GCGGAGCGAT GCCCAACCAG GCGCAGATGC GGATCCTGAA AGAGACGGAGCTGAGGAAGG TGAAGGTGCT TGGATCTGGC GCTTTTGGCA CAGTCTACAA GGGCATCTGGATCCCTGATG GGGAGAATGT GAAAATFCCA GTGGCCATCA AAGTGTTGAG GGAAAACACATCCCCCAAAG CCAACAAAGA AATCTTAGAC GAAGCATACG TGATGGCTGC TGTGGGCTCCCCATATGTCT CCCGCCTTCT GGGCATCTGC CTGACATCCA CGGTGCAGCT GGTGACACAGCTTATGCCCT ATGGCTGCCT CTTAGACCAT GTCCGGGAAA ACCGCGGACG CCTGGGCTCCCAGGACCTGC TGAACTGGTG TATGCAGATT GCCAAGGGGA TGAGCTACCT GGAGGATGTGCGGCTCGTAC ACAGGGACTT GGCCGCTCGG AACGTGCTGG TCAAGAGTCC CAACCATGTCAAAATTACAG ACTTCGGGCT GGCTCGGCTG-3′ (SEQ ID NO:15), and its complement.The template also included a T7 and T3 promoter. The resultant ErbB2specific antisense riboprobe was designated “442 AS.”

[0373] The β-actin riboprobe was synthesized by transcription from a DNAtemplate having the following sequence, 5′-GCTGCCTGAC GGCCAGGTCATCACCATTGG CAATGAGCGG TTCCGCTGCC CTGAGGCACTCTTCCAGCCT TCCTTCCTGGGCATGGAGTC CTGTGGCATC CACGAAACTA CCTTCAACTC CATCATGAAG TGTGACTGTGACATCCGCAA AGACCTGTAC GCCAACACAG TGCTGTCTGG CGGCACCACC ATGTACCCTGGCATTGCCGA CAGGATGCAG AAGGAGATCA CTGCCCTGGC ACCCAGCACA ATGAAGATCAAGATCATTGC TCCTCTGAGC GCAAGTACTC-3′ (SEQ ID NO:16), and its complement.The template included a T3 promoter. The resultant β-actin-specificantisense riboprobe was designated “117 AS.”

[0374] Detection of LIV-1 in Tissue by RNA in Situ Hybridization

[0375] This example provides results from tissue array analyses in whichthe expression of LIV-1 in various tissues was examined. Briefly, LIV-1expression was found to occur in fetal kidney epithelium, developingfetal spinal ganglia including enteric plexuses, fetal brain, adultprostatic epithelium, breast tumors, breast fibroadenomas, lungcarcinoma, squamous lung, colon carcinoma, prostate carcinoma,endometrial carcinoma, ovarian carcinoma, and melanoma.

[0376] Table 4 provides the results of several tissue microarray RNA insitu hybridization analyses. Unless otherwise indicated, the tissuemicroarrays were prepared as described herein. The tabulated results andcomments were generated in studies designated IS2000-060 and IS2000-084in which tissue microarrays were given section numbers merely forinternal reference. The relative expression of LIV-1 is indicated as“+”, “++”, or “+++” for increasing levels of detection, whereas nodetectable expression was indicated as “−.” The LIV-1 riboprobes usedfor RNA in situ hybridization were designated “764 S” as the sense probeand 764 AS” as the antisense probe. Antisense riboprobes specific forErbB2 (probe 442 AS) and β-actin (probe 117 AS, as control) transcriptswere also used in the IS2000-084 study. As used herein, the term “TMA”refers to tissue microarray. The term “NMA” refers to normal tissuemicroarray, where “normal” refers to non-cancerous tissue. TABLE 4Tissue Expression of LIV-1 by RNA in situ Hybridization TissueSection^(c) Probe^(a) Result Comments Study ISH2000-060 Control CellPellet H2000-219.01 764 AS − Control Cell Pellet — 764 S − LIV-1 CellPellet H2000-219.02 764 AS +++ LIV-1 Cell Pellet — 764 S + Various,rhesus & Misc. 02 764 AS − human Various Misc. 02 764 S − 12 wk PlacentaH97-039.02 764 AS − 12 wk Placenta — 764 S − 14.5 wk Fetus H97-106.31764 AS ++ Expression in fetal kidney epithelium, developing spinalganglia, including enteric plexuses 14.5 wk Fetus — 764 S − Fetal BrainH97-045.01 764 AS + Expression over fetal cortical neurons Fetal Brain —764 S − Fetal Brain H97-106.36 764 AS + Adult NMA H2000-165.06 764 AS ++Strong expressioin over prostatic epithelium, focal low level expressionover renal tubules, lung, gall bladder, spleen, heart and pancreasnegative. Adult NMA — 764 S − Breast TumorTMA H2000-94B 764 AS ++Expression elevated in 5 of 13 breast cancers; highly expressed infibroadenomas Tumor Block 3 H2000-165.20 764 AS − Lung Tumor TMAH1999-637 764 AS + Expression in one carcinoma and low level expressionobserved in normal bronchial epithelium Lung Tumor TMA^(b) H2000-27 764AS ++ Poor quality array, 6 cancers show moderate to high expressionColon Tumor TMA H1999-636 764 AS + Relative to breast, low levelexpression in 4 carcinomas Colon Tumor TMA^(b) H2000-26 764 AS − Poorquality array Breast Tumor TMA H1999-635 764 AS ++ Expression observedin 3 carcinomas; highest levels seen in a case of fibroadenoma and oneof selerosing adenosis Prostate Tumor TMA^(b) H2000-25 764 AS ++ Atleast 18 of the cases show reasonable signal; there is a reasonablystrong signal in normal prostate Human NMA H2000-2 764 AS + Low levelsignal over adrenal cortex and renal tubular epithelium Breast NMAHP001595 764 AS ++ Expressed on nipple skin epithelium^(d), breastductal epithelium and acinar epithelium Chimp NMA H2000-185 764 AS +Expression over squamous epithelium Multi-Tumor TMA H2000-132 764 AS +Elevated expression in squamous lung, transitional, endometrial, ovariancarcinomas and melanoma Study IS2000-084 Breast Tumor TMA H2000-94 764AS Positive for LIV-1 mRNA — 442 AS Positive for ErbB2 mRNA — 117 ASPositive for β-actin mRNA # contained samples of pancreas, adrenal,heart, eye, small intestine, kidney, spleen, lymph node, tonsil, skin,breast, lung, brain, colon, liver, aorta, placenta, stomach, ovary,prostate, breast, and skin. All lymph node specimens on H2000-2 wereactin negative suggesting that mRNA was degraded in these samples,whereas heart samples were only weakly actin positive). Breast TMA(section H2000-94B) # contained samples of atypical periductal stromalproliferation (previous excision of cytosarcoma), reactive changes withgiant cells; DCIS (0 of 4 lymph node (LN) positive): Ductal carcinoma,invasive; Ductal carcinoma, poorly-differentiated, invasive (0 of 24 LNpositive); Atypical hyperplasia/CIS: Carcinoma with ductal and lobularfeatures, infiltrative, lymph node metastasis; Ductal carcinoma,invasive, # grade III/III; DCIS, low grade; LCIS (0 of 26 LN pos);Benign breast tissue; Fibroadenoma; Adenocarcinoma, invasive (31 of 31LN pos): Adenosis. Multi tumor TMA (section H2000-132) contained samplesfrom lung tumor (bronchioloalveolarcarcinoma, adenocarcinoma); colonadenocarcinoma; gastric adenocarcinoma; pancreatic ductaladenocarcinoma; heptocellular carcinoma; prostate adenocarcinoma(Gleason grade 2-3): # bladder transitional cell carcinoma; kidneypapillary transitional cell carcinoma: prostate transition cellcarcinoma; renal clear cell carcinoma; endometrial adenocarcinoma; ovarypapillary adenocarcinoma; ovary clear cell adenocarcinoma; lymphoma; andmelanoma.

[0377] As the results of Study IS2000-060 in Table 4 indicate, RNA insitu hybridization confirms elevated expression of LIV-1 in some breastcancers relative to normal breast; expression was elevated in 5 of 13breast cancers in a TMA. High expression was also seen in benign breastdisease, specifically fibroadenomas and sclerosing adenosis. Strongexpression was observed in normal prostatic epithelium as well as inprostate cancers. Expression was seen in epithelium of a number of othertumor types including: squamous lung, transitional, endometrial, ovariancarcinomas and melanoma. With regard to expression in normal tissue,LIV-1 is moderately to highly expressed in normal squamous epithelium(e.g., chimp and human breast skin). Strong expression was observed overnormal prostatic epithelium, focal low level expression over normalrenal tubules. Liver, lung, gall bladder, spleen, heart, and pancreaswere all negative for LIV-1 RNA expression. In fetus, expression wasseen in fetal kidney epithelium as well as in developing spinal ganglia,including enteric plexuses and fetal brain.

[0378] In Study IS2000-084, the relative expression of RNA of P-actin(as control), ErbB2, and LIV-1 in breast tumor were compared. Theresults are shown in Table 4. All samples exhibited adequate expressionof β-actin RNA. Weak to moderate expression of ErbB2 (HER2) RNA was seenin benign and malignant epithelial cells of most cases. Particularlystrong expression was seen in four cases, three cases of infiltratingductal carcinoma, and one case of ductal carcinoma in situ (DCIS). LIV-1RNA was observed in the benign and malignant mammary epithelial cells inmost cases at an intensity ranging from weak to strong, with most casesshowing a moderate level of expression. There was no indication in thetissues sampled for this study that LIV-1 was up-regulated in themalignant cells compared to benign cells.

Example 6 Detecting LIV-1 Expression in Cells

[0379] Diagnosis of breast tumor tissue as the type that overexpressesLIV-1 but does not simultaneously express ErbB2 is useful to allow thephysician to tailor the patients tumor therapy.

[0380] Detection of LIV-1-164647 expression and ErbB2 expression inbreast tumor (or any tumor or other cells) is readily performed bymicroarray technology, as described in Example 1, coupled with ordinaryknowledge of relevant microarray technology procedures.

[0381] Detection of LIV-1-164647 expression in a cell may be performedby in situ hybridization, where the probe for detecting is derived fromthe ECD of LIV-1-164647 and is a cDNA or a RNA having a sequence thathybridizes under stringent conditions to a sequence from nucleotide 412to and including nucleotide 477 of SEQ ID NO:3 or its complementarysequence. Preferably, the probe hybridizes to a sequence from nucleotide446 to and including nucleotide 464 of SEQ ID NO:3 or its complementarysequence. Hybridization protocols useful for this method of detectionare standard in the relevant literature. A non-limiting RNA its usefulhybridization technique useful for detecting LIV-1-164647 expression isdisclosed herein.

[0382] Alternatively, relative expression of LIV-1 and ErbB2 isperformed by contacting an anti-LIV-1-164647 antibody of the inventionthat specifically binds to the extracellular domain of LIV-1-164647protein and comparing the amount of detectable binding with controlcells that do not express LIV-1-164647 protein; with cells (such asSKBR3 cells) that overexpress ErbB2, but do not overexpressLIV-1-164647, and with cells (such as cells expressing LIV-1 or DNA164647) that overexpress LIV-1-164647 but do not overexpress ErbB2,wherein overexpression is determined as at least 1.5-fold greaterexpression in a cell from tumor tissue relative to expression in a cellfrom non-cancerous tissue. The techniques for binding anti-LIV-1antibody and/or anti-ErbB2 antibody are readily determined based ondisclosure provided herein coupled with ordinary skill in the art ofcell surface protein detection. Preferably, the antibody used fordetecting expression of the LIV-1-164647 polypeptide binds to the ECD ofLIV-1-164647, preferably binding to epitope A, epitope B, or epitope Cof an LIV-1 ECD, preferably the LIV-1-164647 ECD. Alternatively theantibody used for detecting expression of an LIV-1 polypeptide binds toa polypeptide comprising an amino acid sequence from amino acid 114 toand including amino acid 135 of SEQ ID NO:4, more preferably comprisinga sequence from amino acid 126 to and including 132 of SEQ ID NO:4.Antibodies useful for practicing this method are described herein andinclude without limitation the monoclonal antibodies produced by one ormore of the hybridomas ATCC ______ (LIV-1.2945.2G1.1C7.2F10); ATCC______ (LIV-1.2982.4A12.1E8.1C4); ATCC ______ (LIV-1.2983.3G9.1D4.1D7);ATCC ______ (LIV-1.2984.6D6.]H10.2C1); ATCC ______(LIV-1.2985.4F3.2D6.1D7); ATCC ______ (LIV-11.2987.1 D8.1C11.2B7); andATCC ______ (LIV-1.2988.1A7.1F2.1H7). Techniques useful for performingantibody binding studies are disclosed herein and are found in therelevant literature.

Example 7 Pharmaceutical Formulations

[0383] Antibodies specifically binding a LIV-1 polypeptide of theinvention, or a fragment of the LIV-1 polypeptide, such as the ECD,which may be identified by the screening assays disclosed herein, can beadministered for the treatment of tumors, including cancers, in the formof pharmaceutical compositions.

[0384] Where antibody fragments are used, the smallest inhibitoryfragment which specifically hinds to the binding domain of the targetprotein is preferred. For example, based upon the variable regionsequences of an antibody, peptide molecules can be designed which retainthe ability to bind the target protein sequence. Such peptides can besynthesized chemically and/or produced by recombinant DNA technology(see, e.g. Marasco et al., Proc. Natl. Acad. Sci. USA 90, 7889-7893[1993]). If the antibody that binds a LIV-1 protein binds to anintracellular portion, and whole antibodies or fragments are used asinhibitors, internalizing the antibodies is preferred. Lipofections orliposomes can be used to deliver the antibody, or an antibody fragment,into cells.

[0385] Therapeutic formulations of the antibodies used in accordancewith the present invention are prepared for storage by mixing anantibody having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride, hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben, catechol;resorcinol, cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysing; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA, sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0386] The formulation herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. For example, it may be desirable to further provideantibodies which bind to vascular endothelial factor (VEGF) in the oneformulation. Alternatively, or in addition, the composition may comprisea cytotoxic agent, cytokine or growth inhibitory agent, provided thatthe cytotoxic agent is other than an anthracycline derivative, e.g.doxorubicin, or epirubicin. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

[0387] The active ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0388] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes.

[0389] Sustained-release preparations may be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped articles, e.g. films, or microcapsules, Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or acetate as aresult of exposure to moisture at 37° C. resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

Example 8 Articles of Manufacture

[0390] In another embodiment of the invention, an article of manufacturecontaining materials useful for the diagnosis or treatment of thedisorders described above is provided. The article of manufacturecomprises a container and a label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for detecting (e.g.diagnosing) or treating the condition and may have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). Theactive agent in the composition is usually an anti-tumor agent capableof interfering with the activity of a gene product identified herein,e.g. an antibody. The label on, or associated with, the containerindicates that the composition is used for diagnosing or treating thecondition of choice. The article of manufacture may further comprise asecond container comprising a pharmaceutically acceptable buffer, suchas phosphate buffered saline, Ringer's solution and dextrose solution.It may further include other materials desirable from a commercial anduser standpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

Example 9 Diagnosis and Prognosis of Tumors

[0391] While cell surface proteins, such as growth receptorsoverexpressed in certain tumors are excellent targets for drugcandidates or tumor (e.g. cancer) treatment, the same proteins alongwith secreted proteins encoded by the genes amplified in tumor cellsfind additional use in the diagnosis and prognosis of tumors. Forexample, antibodies directed against the proteins products of genesamplified in tumor cells can be used as tumor diagnostics orprognostics.

[0392] For example, antibodies, including antibody fragments, can beused to qualitatively or quantitatively detect the expression ofproteins encoded by the amplified genes (“marker gene products”). Theantibody preferably is equipped with a detectable, e.g. fluorescentlabel, and binding can be monitored by light microscopy, flow cytometry,fluorimetry, or other techniques known in the art. These techniques areparticularly suitable, if the amplified gene encodes a cell surfaceprotein, e.g. a growth factor. Such binding assays are performedessentially as described herein.

[0393] In situ detection of antibody binding to the marker gene productscan be performed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a histological specimen is removed fromthe patient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker genie product inthe tissue examined. It will be apparent for those skilled in the artthat a wide variety of histological methods are readily available for insitu detection.

Example 10 Determination of LIV-1 in Tissue or Body Fluid

[0394] Described herein are serological methods for determining thepresence of LIV-1 gene product (e.g. LIV-1-164647 protein or fragmentsthereof) in the body fluid of a mammal, preferably a human patientpotentially suffering from the growth of cells overexpressing LIV-1. Themethod preferably detects the presence and, optionally, quantities, theamount of LIV-1 extracellular domain or fragments thereof in serum ofsuch a patient, thereby providing a relatively non invasive method ofdetecting the overexpression of LIV-1 in a patient.

[0395] Essentially, the processes of this embodiment of the inventioncomprise incubating or otherwise exposing a sample of body fluidpotentially containing LIV-1-164647 extracellular domain or fragmentsthereof, to anti-LIV-1-164647 monoclonal antibodies and detecting thepresence of a reaction product. Those skilled in the art will recognizethat there are many variations of these basic procedures. These include,for example, RIA, ELISA, precipitation, agglutination, complementfixation and immuno-fluorescence. In the currently preferred procedures,the monoclonal antibodies are appropriately labeled for detection.Labels useful in the practice of the invention include, but are notlimited to, moieties, such as enzymes, that must be reacted orderivatized to be detected. The enzyme label can be detected by any ofthe currently utilized calorimetric, spectrophotometric,fluorospectrophotmetric or easometric techniques. The enzyme is combinedwith the antibody with bridging molecules such as carbodimiides,periodate, diisocyanates, glutaraldehyde and the like. Many enzymeswhich can be used in these procedures are known and can be utilized.Examples are peroxidase, alkaline phosphatase. β-glucuronidase.β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase, galactose oxidase plus peroxidase and acid phosphatase.Fluorescent materials which may be used include, for example,fluorescein and its derivatives, rhodamine and its derivatives,auramine, dansyl, umbelliferone, luciferia,2,3-dihydrophthalazinediones, horseradish peroxidase, alkalinephosphatase, lysozyme, and glucose-6-phosphate dehydrogenase. Theantibodies may be tagged with such labels by known methods. Forinstance, coupling agents such as aldehydes, carbodiimides, dimaleimide,imidates, succinimides, bid-diazotized benzadine and the like may beused to tag the antibodies with the above-described fluorescent,chemiluminescent, and enzyme labels. Various labeling techniques aredescribed in Morrison, Methods in Enzymology 32b: 103 [1974]; Syvanen etal., J. Biol. Chem. 284:3762 [1973]; and Bolton and Hunter, Biochem J.133:529 [1973]. Additionally, a radiolabelled antibody can be detectedby any of the currently available counting procedures. Preferred isotopelabels are ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P and ³⁵S.

[0396] Additionally, the following non-limiting assay is useful fordetermining the presence of and to quantitate the amount of specificanti-LIV-1 monoclonal antibody (preferably specific to the extracellulardomain of the LIV-1-164647 gene product, or a portion of theextracellular domain) in a body fluid of a mammal. The body fluid mayinclude, but is not limited to serum, amniotic fluid, milk, umbilicalcord serum, ocular aqueous and vitreous liquids, and ocular vitreousgel.

[0397] Plate Binding Activity Assay Using Humanized Anti-LIV-1Monoclonal Antibody. The method of assaying anti-LIV-1 antibodydescribed herein is meant as an example of such a method and is notmeant to be limiting. A standardized preparation of anti-LIV-1 antibody,preferably specific to the extracellular domain of the LIV-1-167647 geneproduct, controls and serum samples are diluted with Assay Diluent(PBS/0.5% BSA/0.05% Polysorbate 20/0.01% Thimerosal). The dilutions ofstandardized anti-LIV-1 antibody are prepared to span a range ofconcentrations useful for a standard curve. The samples are diluted tofall within the standard curve.

[0398] An aliquot of Coat Antigen in Coating buffer (anti-LIV-1-164647antibody in 0.05 M sodium carbonate buffer) is added to each well of amicrotiter plate and incubated at 2-8° C. for 12-72 hours. The coatingssolution is removed and each well is washed six times with water, thenblotted to remove excess water. An aliquot of Assay Diluent is added toeach well and incubated for 1-2 hours at ambient-temperature withagitation. The wells are washed as in the previous step. Aliquots ofdiluted standard, control and sample solutions are added to the wellsand incubated at ambient temperature for 1 hour with agitation to allowbinding of the antibody to the coating antigen. The wells are washedagain with water as in previous steps.

[0399] Horse radish peroxidase-conjugate (HRP-conjugate, Goat anti-humanIgG Fc conjugated to horseradish peroxidase, (Organon Teknika catalog#55253 or equivalent) is diluted with Assay Diluent to yield anappropriate optical density range between the highest and loweststandards. An aliquot of the HRP-conjugate solution is added to eachwell and incubated at ambient temperature for 1 hour with agitation. Thewells are washed with water as in previous steps.

[0400] An aliquot of Substrate Solution (o-phenylenediamine (OPD) 5 mgtablet (Sigma P6912 or equivalent) in 12.5 ml 4 mM H₂O₂ in PBS) is addedto each well and incubated for a sufficient period of time(approximately 8-10 minutes) in the dark at ambient temperature to allowcolor development. The reaction is stopped with an aliquot of 4.5 Nsulfuric acid. Optical density is read at 490-492 nm for detectionabsorbance and 405 nm for reference absorbance. The standard curve dataare plotted and the results for the controls and samples are determinedfrom the standard curve.

Example 11 Methods of Treatment

[0401] It is contemplated that, according to the present invention, theanti-LIV-1 antibodies or other LIV-1 activity-blocking molecules may beused to treat various conditions characterized by overexpression and/oractivation of the LIV-1 gene product with or without coexpression ofErbB2 above the ErbB2 expression found in healthy, nonmalignant cells,Exemplary conditions or disorders to be treated with such antibodies andother compounds, including, but not limited to, small organic andinorganic molecules, peptides, antisense molecules, etc. include benignor malignant tumors (e.g. breast, prostate, lung, and colon, as well asrenal, liver, kidney, bladder, gastric, ovarian, colorectal, pancreatic,vulval, thyroid, hepatic carcinomas; sarcomas; glioblastomas, andvarious head and neck tumors); leukemias and lymphoid malignancies;other disorders such as neuronal, glial, astrocytal, hypothalamic andother glandular, macrophagal, epithelial, stromal and blastocoelicdisorders, and inflammatory, angiogenic and immunologic disorders. Wherean antibody is used to treat an LIV-1 overexpression related disorder,the antibody is preferably an anti-LIV-1-164647 antibody, morepreferably a humanized antibody which binds to a polypeptide comprisingan amino acid sequence from amino acid 114 to and including amino acid135 of SEQ ID NO:4.

[0402] The anti-tumor agents of the present invention, e.g. antibodies,are administered to a mammal, preferably a human, in accord with knownmethods, such as intravenous administration as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. Intravenousadministration of the antibody is preferred.

[0403] Other therapeutic regimens may be combined with theadministration of the anti-cancer agents, e.g. antibodies of the instantinvention. For example, the patient to be treated with such anti-canceragents may also receive radiation therapy. Alternatively, or inaddition, a chemotherapeutic agent may be administered to the patient.Preparation and dosing schedules for such chemotherapeutic agents may beused according to manufacturers' instructions or as determinedempirically by the skilled practitioner. Preparation and dosingschedules for such chemotherapy are also described in ChemotherapyService Ed. M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992). Thechemotherapeutic agent may precede, or follow administration of theanti-tumor agent, e.g. antibody, or may be given simultaneouslytherewith. The antibody may be combined with an anti-oestrogen compoundsuch as tamoxifen or an anti-progesterone such as onapristone (see, EP616812) in dosages known for such molecules.

[0404] It may be desirable to also administer antibodies against othertumor associated antigens, such as antibodies which bind to the ErbB2,EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF).Alternatively, or in addition, two or more antibodies binding the sameor two or more different antigens disclosed herein may beco-administered to the patient. Sometimes, it may be beneficial to alsoadminister one or more cytokines to the patient. In a preferredembodiment, the antibodies herein are co-administered with a growthinhibitory agent. For example, the growth inhibitory agent may beadministered first, followed by an antibody of the present invention.However, simultaneous administration or administration of the antibodyof the present invention first is also contemplated. Suitable dosagesfor the growth inhibitory agent are those presently used and may belowered due to the combined action (synergy) of the growth inhibitoryagent and the antibody herein.

[0405] Where combined administration of a chemotherapeutic agent isdesired, the combined administration includes co-administration ineither order, wherein preferably there is a time period while both (orall) active agents simultaneously exert their biological activities.Preparation and dosing schedules for such chemotherapeutic agents may beused according to manufacturers instructions or as determinedempirically by the skilled practitioner. Preparation and dosingschedules for such chemotherapy are also described in ChemotherapyService Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992). Thechemotherapeutic agent may precede, or follow administration of theantibody or may be given simultaneously therewith. The antibody may becombined with an anti-estrogen compound such as tamoxifen or ananti-progesterone such as onapristone (see, EP 616812) in dosages knownfor such molecules.

[0406] It may be desirable to also administer antibodies against othertumor associated antigens, such as antibodies which bind to vascularendothelial factor (VEGF). Alternatively, or in addition, two or moreanti-LIV-1 antibodies may be co-administered to the patient. Sometimes,it may be beneficial to also administer one or more cytokines to thepatient. The anti-LIV-1 antibody may be co-administered with a growthinhibitory agent. For example, the growth inhibitory agent may beadministered first, followed by the anti-LIV-1 antibody, However,simultaneous administration or administration of the anti-LIV-1 antibodyfirst is also contemplated. Suitable dosages for the growth inhibitoryagent are those presently used and may be lowered due to the combinedaction (synergy) of the growth inhibitory agent and anti LIV-1 antibody.

[0407] In addition to the above therapeutic regimens, the patient may besubjected to surgical removal of cancer cells and/or radiation therapy.

[0408] For example, depending on the type and severity of the disease,about 1 μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of antibody is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

Example 12 Antibody Binding Studies

[0409] The results of the gene amplification study can be furtherverified by antibody binding studies, in which the ability ofanti-LIV-1-164647 antibodies to detect the presence of or to inhibit theeffect of the LIV-1 polypeptides on tumor (cancer) cells is tested.Exemplary antibodies include polyclonal, monoclonal, humanized,bispecific, and heteroconjugate antibodies, the preparation of whichwill be described hereinbelow.

[0410] Antibody binding studies may be carried out in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays, Zola, Monoclonal Antibodies: AManual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

[0411] Competitive binding assays rely on the ability of a labeledstandard to compete with the test sample analyte for binding with alimited amount of antibody. The amount of target protein (encoded by agene amplified in a tumor cell) in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies preferably are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

[0412] Sandwich assays involve the use of two antibodies, each capableof binding to a different immunogenic portion, or epitope, of theprotein to be detected. In a sandwich assay, the test sample analyte isbound by a first antibody which is immobilized on a solid support, andthereafter a second antibody binds to the analyte, thus forming aninsoluble three-part complex. See, e.g. U.S. Pat. No. 4.376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

[0413] For immunohistochemistry, the tumor sample may be fresh or frozenor may be embedded in paraffin and fixed with a preservative such asformalin, for example.

Example 13 Cell-Based Tumor Assays

[0414] Cell-based assays and animal models for tumors (e.g. cancers) canbe used to verify the findings of the gene amplification assay, andfurther understand the relationship between the genes identified hereinand the development and pathogenesis of neoplastic cell growth. The roleof gene products identified herein in the development and pathology oftumor or cancer can be tested by using primary tumor cells or cellslines that have been identified to amplify the genes herein. Such cellsinclude, for example, the breast and prostate cancer cells and celllines listed above.

[0415] In a different approach, cells of a cell type known to beinvolved in a particular tumor are transfected with the cDNAs herein,and the ability of these cDNAs to induce excessive growth is analyzed.Suitable cells include, for example, stable tumor cells lines such as,the B104-1-1 cell line (stable NIH-3T3 cell line transfected with theneu protooncogene) and ras-transfected NIH-3T3 cells, which can betransfected with the desired gene, and monitored for tumorogenic growth.Such transfected cell lines can then be used to test the ability ofpoly- or monoclonal antibodies or antibody compositions to inhibittumorogenic cell growth by exerting cytostatic or cytotoxic activity onthe growth of the transformed cells, or by mediating antibody-dependentcellular cytotoxicity (ADCC). Cells transfected with the codingsequences of the genes identified herein can further be used to identifydrug candidates for the treatment of cancer.

[0416] In addition, primary cultures derived from tumors in transgenicanimals (as described below) can be used in the cell-based assaysherein, although stable cell lines are preferred. Techniques to derivecontinuous cell lines from transgenic animals are well known in the art(see, e.g. Small et al., Mol. Cell. Biol. 5, 642-648 [1985]).

Example 14 Animal Models

[0417] A variety of well known animal models can be used to furtherunderstand the role of the genes identified herein in the developmentand pathogenesis of tumors, and to test the efficacy of candidatetherapeutic agents, including antibodies, and other antagonists of thenative polypeptides, including small molecule antagonists. The in vivonature of such models makes them particularly predictive of responses inhuman patients. Animal models of tumors and cancers (e.g. breast cancer,colon cancer, prostate cancer, lung cancer, etc.) include bothnon-recombinant and recombinant (transgenic) animals. Non-recombinantanimal models include, for example, rodent, e.g., murine models. Suchmodels can be generated by introducing tumor cells into syngeneic miceusing standard techniques, e.g. subcutaneous injection, tail veininjection, spleen implantation, intraperitoneal implantation,implantation under the renal capsule, or orthopin implantation, e.g.colon cancer cells implanted in colonic tissue. (See, e.g. PCTpublication No. WO 97/33551, published Sep. 18, 1997).

[0418] Probably the most often used animal species in oncologicalstudies are immunodeficient mice and, in particular, nude mice. Theobservation that the nude mouse with hypo/aplasia could successfully actas a host for human tumor xenografts has lead to its widespread use forthis purpose. The autosomal recessive nu gene has been introduced into avery large number of distinct congenic strains of nude mouse, including,for example, ASW, A/He, AKR, BALB/c, B10.LP, C17, C3H. C57BL, C57, CBA,DBA, DDD, I/st, NC, NFR, NFS, NFS/N, NZB, NZC, NZW, P, RIII and SJL. Inaddition, a wide variety of other animals with inherited immunologicaldetects other than the nude mouse have been bred and used as recipientsof tumor xenografts. For further details see, e.g. The Nude Mouse inOncology Research, E. Boven and B. Winograd, eds., CRC Press, Inc. 1991.

[0419] The cells introduced into such animals can be derived from knowntumor/cancer cell lines, such as, any of the above-listed tumor celllines, and, for example, the B104-1-1 cell line (stable NIH-3T3 cellline transfected with the neu protooncogene); ras-transfected NIH-3T3cells; Caco-2 (ATCC HTB-37); a moderately well-differentiated grade 11human colon adenocarcinoma cell line, HT-29 (ATCC HTB-38), or fromtumors and cancers. Samples of tumor or cancer cells can be obtainedfrom patients undergoing surgery, using standard conditions, involvingfreezing and storing in liquid nitrogen (Karmali et al., Br. J. Cancer48, 689-696 [1983]). Tumor cells can be introduced into animals, such asnude mice, by a variety of procedures. The subcutaneous (s.c.) space inmice is very suitable for tumor implantation. Tumors can be transplanteds.c. as solid blocks, as needle biopsies by use of a trochar, or as cellsuspensions. For solid block or trochar implantation, tumor tissuefragments of suitable size are introduced into the s.c. space. Cellsuspensions are freshly prepared from primary tumors or stable tumorcell lines, and injected subcutaneously. Tumor cells can also beinjected as subdermal implants. In this location, the inoculum isdeposited between the lower part of the dermal connective tissue and thes.c. tissue. Boven and Winograd (1991), supra.

[0420] Animal models of breast cancer can be generated, for example, byimplanting rat neuroblastoma cells (from which the lieu oncogen wasinitially isolated), or neu-transformed NIH-3T3 cells into nude mice,essentially as described by Drebin et al. PNAS USA 83, 9129-9133 (1986).

[0421] Similarly, animal models of colon cancer can be generated bypassaging colon cancer cells in animals, e.g. nude mice, leading to theappearance of tumors in these animals. An orthotopic transplant model ofhuman colon cancer in nude mice has been described, for example, by Wanget al., Cancer Research 54, 4726-4728 (1994) and Too et al., CancerResearch 55, 681-684 (1995). This model is based on the so-called“METAMOUSE™” sold by AntiCancer, Inc. (San Diego, Calif.).

[0422] Tumors that arise in animals can be removed and cultured invitro. Cells from the in vitro cultures can then be passaged to animals.Such tumors can serve as targets for further testing or drug screening.Alternatively, the tumors resulting from the passage can be isolated andRNA from pre-passage cells and cells isolated after one or more roundsof passage analyzed for differential expression of genes of interest.Such passaging techniques can be performed with any known tumor orcancer cell lines.

[0423] For example, Meth A. CMS4, CMS5, CMS21, and WEHI-164 arechemically induced fibrosarcomas of BALB/c female mice (DeLco et al., J.Exp. Med. 146, 720 [1977]), which provide a highly controllable modelsystem for studying the anti-tumor activities of various agents(Palladino et al., J. Immunol. 138, 4023-4032 [1987]). Briefly, tumorcells are propagated in vitro in cell culture. Prior to injection intothe animals, the cell lines are washed and suspended in buffer, at acell density of about 1×10⁶ to 10×10⁷ cells/ml. The animals are theninfected subcutaneously with 10 to 100 μl of the cell suspension,allowing one to three weeks for a tumor to appear.

[0424] In addition, the Lewis lung (3LL) carcinoma of mice, which is oneof the most thoroughly studied experimental tumors, can be used as aninvestigational tumor model. Efficacy in this tumor model has beencorrelated with beneficial effects in the treatment of human patientsdiagnosed with small cell carcinoma of the lung (SCCL). This tumor canbe introduced in normal mice upon injection of tumor fragments from anaffected mouse or of cells maintained in culture (Zupi et al., Br. J.Cancer 41, suppl, 4, 309 [1980]), and evidence indicates that tumors canbe started from injection of even a single cell and that a very highproportion of infected tumor cells survive. For further informationabout this tumor model see Zacharski, Haemostasis 16, 300-320 [1986]).

[0425] One way of evaluating the efficacy of a test compound on animplanted tumor in an animal model is to measure the size of the tumorbefore and after treatment. Traditionally, the size of implanted tumorshas been measured with a slide caliper in two or three dimensions. Themeasure limited to two dimensions does not accurately reflect the sizeof the tumor, therefore, it is usually converted into the correspondingvolume by using a mathematical formula. However, the measurement oftumor size is very inaccurate. The therapeutic effects of a drugcandidate can be better described as treatment-induced growth delay andspecific growth delay. Another important variable in the description oftumor growth is the tumor volume doubling time. Computer programs forthe calculation and description of tumor growth are also available, suchas the program reported by Rygaard and Spang-Thomsen, Proc. 6td Int.Workshop of Immune-Deficient Animals, Wu and Sheng eds., Basel. 1989,301. It is noted, however, that necrosis and inflammatory responsesfollowing treatment may actually result in an increase in tumor size, atleast initially. Therefore, these changes need to be carefullymonitored, by a combination of a morphometric method and flow cytometricanalysis.

[0426] Recombinant (transgenic) animal models can be engineered byintroducing the coding portion of the genes identified herein into thegenome of animals of interest, using standard techniques for producingtransgenic animals. Animals that can serve as a target for transgenicmanipulation include, without limitation, mice, rats, rabbits, guineapigs, sheep, goats, pigs, and non-human primates, e.g. baboons,chimpanzees and monkeys. Techniques known in the art to introduce atransgene into such animals include pronucleic microinjection (Hoppe andWanger. U.S. Pat. No. 4,873,191); retrovirus-mediated gene transfer intogerm lines (e.g. Van der Putten et al., Proc. Natl. Acad. Sci. USA 82,6148-615 [1985]); gene targeting in embryonic stem cells (Thompson etal., Cell 56, 313-321 [1989]); electroporation of embryos (Lo, Mol.Cel., Biol. 3, 1803-1814 [1983]); sperm-mediated gene transfer(Lavitrano et al., Cell 57, 717-73 [1989]). For review, see, forexample, U.S. Pat. No. 4,736,866.

[0427] For the purpose of the present invention, transgenic animalsinclude those that carry the transgene only in part of their cells(“mosaic animals”). The transgene can be integrated either as a singletransgene, or in concatamers, e.g., head-to-head or head-to-tailtandems. Selective introduction of a transgenic into a particular celltype is also possible by following, for example, the technique of Laskoet al., Proc. Natl. Acad. Sci. USA 89, 6232-636(1992).

[0428] The expression of the transgene in transgenic animals can bemonitored by standard techniques. For example, Southern blot analysis orPCR amplification can be used to verify the integration of thetransgene. The level of mRNA expression can then be analyzed usingtechniques such as in situ hybridization. Northern blot analysis, PCR,or immunocytochemistry. The animals are further examined for signs oftumor or cancer development.

[0429] Alternatively “knock out” animals can be constructed high have adefective or altered gene encodings a LIV-1-164647 polypeptideidentified herein, as a result of homologous recombination between theendogenous gene encoding the polypeptide and altered genomic DNAencoding the same polypeptide introduced into an embryonic cell of theanimal. For example, cDNA encoding) a particular LIV-1 polypeptide canbe used to clone genomic DNA encoding that polypeptide in accordancewith established techniques. A portion of the genomic DNA encoding aparticular LIV-1 polypeptide can be deleted or replaced with anothergene, such as a (gene encoding a selectable marker which can be used tomonitor integration. Typically, several kilobases of unaltered linkingDNA (both at the 5′ and 3′ ends) are included in the vector [see e.g.,Thomas and Capecchi, Cell, 51:503(1987) for a description of homologousrecombination vectors]. The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected[see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat to formaggregation chimeras [see e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knockout animals can becharacterized for instance, by their ability to defend against certainpathological conditions and by their development of pathologicalconditions due to absence of the LIV-1 polypeptide.

[0430] The efficacy of antibodies specifically binding the polypeptidesidentified herein and other drug candidates, can be tested also in thetreatment of spontaneous animal tumors. A suitable target for suchstudies is the feline oral squamous cell carcinoma (SCC). Feline oralSCC is a highly invasive, malignant tumor that is the most common oralmalignancy of cats, accounting for over 60% of the oral tumors reportedin this species. It rarely metastasizes to distant sites, although thislow incidence of metastasis may merely be a reflection of the shortsurvival times for cats with this tumor. These tumors are usually notamenable to surgery, primarily because of the anatomy of the feline oralcavity. At present, there is no effective treatment for this tumor.Prior to entry into the study, each cat undergoes complete clinicalexamination, biopsy, and is scanned by computed tomography, Catsdiagnosed with sublingual oral squamous cell tumors are excluded fromthe study. The tongue can become paralyzed as a result of such tumor,and even if the treatment kills the tumor, the animals may not be ableto feed themselves. Each cat is treated repeatedly, over a longer periodof time. Photographs of the tumors will be taken daily during thetreatment period, and at each subsequent recheck. After treatment, eachcat undergoes another computed tomography scan. Computed tomographyscans and thoracic radiograms are evaluated every 8 weeks thereafter.The data are evaluated for differences in survival, response andtoxicity as compared to control groups. Positive response may requireevidence of tumor regression, preferably with improvement of quality oflife and/or increased life span.

[0431] In addition, other spontaneous animal tumors, such asfibrosarcoma, adenocarcinoma, lymphoma, chrondroma, leiomyosarcoma ofdogs, cats, and baboons can also be tested. Of these mammaryadenocarcinoma in dogs and cats is a preferred model as its appearanceand behavior are very similar to those in humans. However, the use ofthis model is limited by the rare occurrence of this type of tumor inanimals.

Example 15 Screening Assays for Drug Candidates

[0432] Screening assays for drug candidates are designed to identifycompounds that bind or complex with the polypeptides encoded by thegenes identified herein, or otherwise interfere with the interaction ofthe encoded polypeptides with other cellular proteins. Such screeningassays will include assays amenable to high-throughput screening ofchemical libraries, making them particularly suitable for identifyingsmall molecule drug candidates. Small molecules contemplated includesynthetic organic or inorganic compounds, including peptides, preferablysoluble peptides, (poly)peptide-immunoglobulin fusions, and, inparticular, antibodies including, without limitation, poly- andmonoclonal antibodies and antibody fragments, single-chain antibodies,anti-idiotypic antibodies, and chimeric or humanized versions of suchantibodies or fragments, as well as human antibodies and antibodyfragments. The assays can be performed in a variety of formats,including protein-protein binding assays, biochemical screening assays,immunoassays and cell based assays, which are well characterized in theart.

[0433] All assays are common in that they call for contacting the drugcandidate with a polypeptide encoded by a nucleic acid identified hereinunder conditions and for a time sufficient to allow these two componentsto interact.

[0434] In binding assays, the interaction is binding and the complexformed can be isolated or detected in the reaction mixture. In aparticular embodiment, the polypeptide encoded by the gene identifiedherein or the drug candidate is immobilized on a solid phase, e.g. on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the polypeptide and drying. Alternatively, an immobilizedantibody, e.g. a monoclonal antibody, specific for the polypeptide to beimmobilized can be used to anchor it to a solid surface. The assay isperformed by adding the non-immobilized component, which may be labeledby a detectable label, to the immobilized component, e.g. the coatedsurface containing the anchored component. When the reaction iscomplete, the non-reacted components are removed, e.g. by washing, andcomplexes anchored on the solid surface are detected. When theoriginally non-immobilized component carries a detectable label, thedetection of label immobilized on the surface indicates that complexingoccurred. Where the originally non-immobilized component does not carrya label, complexing can be detected, for example, by using a labeledantibody specifically binding the immobilized complex.

[0435] If the candidate compound interacts with but does not bind to aparticular LIV-1 polypeptide encoded by a nucleic acid sequencedescribed herein, its interaction with that polypeptide can be assayedby methods well known for detecting protein-protein interactions. Suchassays include traditional approaches, such as, cross-linkingco-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers [Fields and Song, Nature (London) 340, 245-246 (1989);Chien et al., Proc. Natl Acad. Sci. USA 88, 9578-9582 (1991)] asdisclosed by Chevray and Nathans [Proc. Natl. Acad. Sci. USA 89,5789-5793 (1991)]. Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA binding domain, while the other one functioning as the transcriptionactivation domain. The yeast expression system described in theforegoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

[0436] Compounds that interfere with the interaction of a LIV-1-encodinggene identified herein and other intra- or extracellular components canbe tested as follows: usually a reaction mixture is prepared containingthe product of the amplified gene and the intra- or extracellularcomponent under conditions and for a time allowing for the interactionand binding of the two products. To test the ability of a test compoundto inhibit binding, the reaction is run in the absence and in thepresence of the test compound. In addition, a placebo may be added to athird reaction mixture, to serve as positive control. The binding(complex formation) between the test compound and the intra orextracellular component present in the mixture is monitored as describedhereinabove. The formation of a complex in the control reaction(s) butnot in the reaction mixture containing the test compound indicates thatthe test compound interferes with the interaction of the test compoundand its reaction partner.

Example 16 Other Compositions and Methods for the Treatment of Tumors

[0437] The compositions useful in the treatment of tumors associatedwith the amplification of the genes identified herein include, withoutlimitation, antibodies, small organic and inorganic molecules, peptides,phosphopeptides, antisense and ribozyme molecules, triple helixmolecules, etc. that inhibit the expression and/or activity of thetarget gene product.

[0438] For example, antisense RNA and RNA molecule act to directly blockthe translation of mRNA by hybridizing to targeted mRNA and preventingprotein translation. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation initiation site,e.g. between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

[0439] Ribozymes are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA. Ribozymes act by sequence-specifichybridization to the complementary target RNA, followed byendonucleolytic cleavage. Specific ribozyme cleavage sites within apotential RNA target can be identified by known techniques. For furtherdetails see, e.g. Rossi, Current Biology 4, 469-471 (1994), and PCTpublication No. WO 97/33551 (published Sep. 18, 1997).

[0440] Nucleic acid molecules in triple helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple helix formation via Hoogsteen basepairing rules, which generally require sizeable stretches of purines orpyrimidines on one strand of a duplex. For further details see, e.g. PCTpublication No. WO 97/33551, supra

[0441] These molecules can be identified by any or any combination ofthe screening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

[0442] Deposit of Material

[0443] The following material has been deposited with the American TypeCulture Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209, USA (ATCC): Material ATCC Dep No. Deposit Date Plasmid;DNA164647 1803-1 PTA-1534 Mar. 21, 2000 Hybridomas;LIV-1.2945.2G1.1C7.2F10 PTA-2961 Jan. 23, 2001 LIV-1.2982.4A12.1E8.1C4PTA-2962 Jan. 23, 2001 LIV-1.2983.3G9 1D4.1D7 PTA-2963 Jan. 23, 2001LIV-1.2984.6D6.1H10.2C1 PTA-2964 Jan. 23, 2001 LIV-1.2985 4F3.2D6 1D7PTA-2960 Jan. 23, 2001 LIV-1.2987 1D8.1C11.2B7 PTA-2959 Jan. 23, 2001LIV-1.2988 1A7.1F2.1H7 PTA-2965 Jan. 23, 2001

[0444] This deposit was made under the provisions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe purpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon influence of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trade to be entitled theretoaccording to 35 U.S.C. §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 886 OG 638)

[0445] The assignee of the present application has agreed that it aculture of the materials on deposit should die or be lost or destroyedwhen cultivated under suitable conditions, the materials will bepromptly replaced on notification with another of the same. Availabilityof the deposited material is not to be construed as a license topractice the invention in contravention of the right granted under theauthority of any government in accordance with its patent laws.

[0446] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.The present invention is not to be limited in scope by the constructdeposited, since the deposited embodiment is intended as a singleillustration of certain aspects of the invention and any constructs thatare functionally equivalent are within the scope of the this invention.The deposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.The disclosures of all citations in the specification are expresslyincorporated herein by reference.

1 19 1 3461 DNA Homo sapien 1 ctcgtgccga attcggcacg agaccgcgtgttcgcgcctg gtagagattt 50 ctcgaagaca ccagtgggcc cgtgtggaac caaacctgcgcgcgtggccg 100 ggccgtggga caacgaggcc gcggagacga aggcgcaatg gcgaggaagt150 tatctgtaat cttgatcctg acctttgccc tctctgtcac aaatcccctt 200catgaactaa aagcagctgc tttcccccag accactgaga aaattagtcc 250 gaattgggaatctggcatta atgttgactt ggcaatttcc acacggcaat 300 atcatctaca acagcttttctaccgctatg gagaaaataa ttctttgtca 350 gttgaagggt tcagaaaatt acttcaaaatataggcatag ataagattaa 400 aagaatccat atacaccatg accacgacca tcactcagaccacgagcatc 450 actcagacca tgagcgtcac tcagaccatg agcatcactc agaccacgag500 catcactctg accataatca tgctgcttct ggtaaaaata agcgaaaagc 550tctttgccca gaccatgact cagatagttc aggtaaagat cctagaaaca 600 gccaggggaaaggagctcac cgaccagaac atgccagtgg tagaaggaat 650 gtcaaggaca gtgttagtgctagtgaagtg acctcaactg tgtacaacac 700 tgtctctgaa ggaactcact ttctagagacaatagagact ccaagacctg 750 gaaaactctt ccccaaagat gtaagcagct ccactccacccagtgtcaca 800 tcaaagagcc gggtgagccg gctggctggt aggaaaacaa atgaatctgt850 gagtgagccc cgaaaaggct ttatgtattc cagaaacaca aatgaaaatc 900ctcaggagtg tttcaatgca tcaaagctac tgacatctca tggcatgggc 950 atccaggttccgctgaatgc aacagagttc aactatctct gtccagccat 1000 catcaaccaa attgatgctagatcttgtct gattcataca agtgaaaaga 1050 aggctgaaat ccctccaaag acctattcattacaaatagc ctgggttggt 1100 ggttttatag ccatttccat catcagtttc ctgtctctgctgggggttat 1150 cttagtgcct ctcatgaatc gggtgttttt caaatttctc ctgagtttcc1200 ttgtggcact ggccgttggg actttgagtg gtgatgcttt tttacacctt 1250cttccacatt ctcatgcaag tcaccaccat agtcatagcc atgaagaacc 1300 agcaatggaaatgaaaagag gaccactttt cagtcatctg tcttctcaaa 1350 acatagaaga aagtgcctattttgattcca cgtggaaggg tctaacagct 1400 ctaggaggcc tgtatttcat gtttcttgttgaacatgtcc tcacattgat 1450 caaacaattt aaagataaga agaaaaagaa tcagaagaaacctgaaaatg 1500 atgatgatgt ggagattaag aagcagttgt ccaagtatga atctcaactt1550 tcaacaaatg aggagaaagt agatacagat gatcgaactg aaggctattt 1600acgagcagac tcacaagagc cctcccactt tgattctcag cagcctgcag 1650 tcttggaagaagaagaggtc atgatagctc atgctcatcc acaggaagtc 1700 tacaatgaat atgtacccagagggtgcaag aataaatgcc attcacattt 1750 ccacgataca ctcggccagt cagacgatctcattcaccac catcatgact 1800 accatcatat tctccatcat caccaccacc aaaaccaccatcctcacagt 1850 cacagccagc gctactctcg ggaggagctg aaagatgccg gcgtcgccac1900 tttggcctgg atggtgataa tgggtgatgg cctgcacaat ttcagcgatg 1950gcctagcaat tggtgctgct tttactgaag gcttatcaag tggtttaagt 2000 acttctgttgctgtgttctg tcatgagttg cctcatgaat taggtgactt 2050 tgctgttcta ctaaaggctggcatgaccgt taagcaggct gtcctttata 2100 atgcattgtc agccatgctg gcgtatcttggaatggcaac aggaattttc 2150 attggtcatt atgctgaaaa tgtttctatg tggatatttgcacttactgc 2200 tggcttattc atgtatgttg ctctggttga tatggtacct gaaatgctgc2250 acaatgatgc tagtgaccat ggatgtagcc gctgggggta tttcttttta 2300cagaatgctg ggatgctttt gggttttgga attatgttac ttattccata 2350 tttgaacataaaatcgtgtt cgtataaatt tctagttaag gtttaaatgc 2400 tagagtagct taaaaagttgtcatagtttc agtaggtcat agggagatga 2450 gtttgtatgc tgtactatgc agcgtttaaagttagtgggt tttgtgattt 2500 ttgtattgaa tattgctgtc tgttacaaag tcagttaaaggtacgtttta 2550 atatttaagt tattctatct tggagataaa atctgtatgt gcaattcacc2600 ggtattacca gtttattatg taaacaagag atttggcatg acatgttctg 2650tatgtttcag ggaaaaatgt ctttaatgct ttttcaagaa ctaacacagt 2700 tattcctatactggatttta ggtctctgaa gaactgctgg tgtttaggaa 2750 taagaatgtg catgaagcctaaaataccaa gaaagcttat actgaattta 2800 agcaaagaaa taaaggagaa aagagaagaatctgagaatt ggggaggcat 2850 agattcttat aaaaatcaca aaatttgttg taaattagaggggagaaatt 2900 tagaattaag tataaaaagg cagaattagt atagagtaca ttcattaaac2950 atttttgtca ggattatttc ccgtaaaaac gtagtgagca ctctcatata 3000ctaattagtg tacatttaac tttgtataat acagaaatct aaatatattt 3050 aatgaattcaagcaatatac acttgaccaa gaaattggaa tttcaaaatg 3100 ttcgtgcggg ttatataccagatgagtaca gtgagtagtt tatgtatcac 3150 cagactgggt tattgccaag ttatatatcaccaaaagctg tatgactgga 3200 tgttctggtt acctggttta caaaattatc agagtagtaaaactttgata 3250 tatatgagga tattaaaact acactaagta tcatttgatt cgattcagaa3300 agtactttga tatctctcag tgcttcagtg ctatcattgt gagcaattgt 3350ctttatatac ggtactgtag ccatactagg cctgtctgtg gcattctcta 3400 gatgtttcttttttacacaa taaattcctt atatcagctt gaaaaaaaaa 3450 aaaaaaaaaa a 3461 2 752PRT Homo sapien 2 Met Ala Arg Lys Leu Ser Val Ile Leu Ile Leu Thr PheAla Leu 1 5 10 15 Ser Val Thr Asn Pro Leu His Glu Leu Lys Ala Ala AlaPhe Pro 20 25 30 Gln Thr Thr Glu Lys Ile Ser Pro Asn Trp Glu Ser Gly IleAsn 35 40 45 Val Asp Leu Ala Ile Ser Thr Arg Gln Tyr His Leu Gln Gln Leu50 55 60 Phe Tyr Arg Tyr Gly Glu Asn Asn Ser Leu Ser Val Glu Gly Phe 6570 75 Arg Lys Leu Leu Gln Asn Ile Gly Ile Asp Lys Ile Lys Arg Ile 80 8590 His Ile His His Asp His Asp His His Ser Asp His Glu His His 95 100105 Ser Asp His Glu Arg His Ser Asp His Glu His His Ser Asp His 110 115120 Glu His His Ser Asp His Asn His Ala Ala Ser Gly Lys Asn Lys 125 130135 Arg Lys Ala Leu Cys Pro Asp His Asp Ser Asp Ser Ser Gly Lys 140 145150 Asp Pro Arg Asn Ser Gln Gly Lys Gly Ala His Arg Pro Glu His 155 160165 Ala Ser Gly Arg Arg Asn Val Lys Asp Ser Val Ser Ala Ser Glu 170 175180 Val Thr Ser Thr Val Tyr Asn Thr Val Ser Glu Gly Thr His Phe 185 190195 Leu Glu Thr Ile Glu Thr Pro Arg Pro Gly Lys Leu Phe Pro Lys 200 205210 Asp Val Ser Ser Ser Thr Pro Pro Ser Val Thr Ser Lys Ser Arg 215 220225 Val Ser Arg Leu Ala Gly Arg Lys Thr Asn Glu Ser Val Ser Glu 230 235240 Pro Arg Lys Gly Phe Met Tyr Ser Arg Asn Thr Asn Glu Asn Pro 245 250255 Gln Glu Cys Phe Asn Ala Ser Lys Leu Leu Thr Ser His Gly Met 260 265270 Gly Ile Gln Val Pro Leu Asn Ala Thr Glu Phe Asn Tyr Leu Cys 275 280285 Pro Ala Ile Ile Asn Gln Ile Asp Ala Arg Ser Cys Leu Ile His 290 295300 Thr Ser Glu Lys Lys Ala Glu Ile Pro Pro Lys Thr Tyr Ser Leu 305 310315 Gln Ile Ala Trp Val Gly Gly Phe Ile Ala Ile Ser Ile Ile Ser 320 325330 Phe Leu Ser Leu Leu Gly Val Ile Leu Val Pro Leu Met Asn Arg 335 340345 Val Phe Phe Lys Phe Leu Leu Ser Phe Leu Val Ala Leu Ala Val 350 355360 Gly Thr Leu Ser Gly Asp Ala Phe Leu His Leu Leu Pro His Ser 365 370375 His Ala Ser His His His Ser His Ser His Glu Glu Pro Ala Met 380 385390 Glu Met Lys Arg Gly Pro Leu Phe Ser His Leu Ser Ser Gln Asn 395 400405 Ile Glu Glu Ser Ala Tyr Phe Asp Ser Thr Trp Lys Gly Leu Thr 410 415420 Ala Leu Gly Gly Leu Tyr Phe Met Phe Leu Val Glu His Val Leu 425 430435 Thr Leu Ile Lys Gln Phe Lys Asp Lys Lys Lys Lys Asn Gln Lys 440 445450 Lys Pro Glu Asn Asp Asp Asp Val Glu Ile Lys Lys Gln Leu Ser 455 460465 Lys Tyr Glu Ser Gln Leu Ser Thr Asn Glu Glu Lys Val Asp Thr 470 475480 Asp Asp Arg Thr Glu Gly Tyr Leu Arg Ala Asp Ser Gln Glu Pro 485 490495 Ser His Phe Asp Ser Gln Gln Pro Ala Val Leu Glu Glu Glu Glu 500 505510 Val Met Ile Ala His Ala His Pro Gln Glu Val Tyr Asn Glu Tyr 515 520525 Val Pro Arg Gly Cys Lys Asn Lys Cys His Ser His Phe His Asp 530 535540 Thr Leu Gly Gln Ser Asp Asp Leu Ile His His His His Asp Tyr 545 550555 His His Ile Leu His His His His His Gln Asn His His Pro His 560 565570 Ser His Ser Gln Arg Tyr Ser Arg Glu Glu Leu Lys Asp Ala Gly 575 580585 Val Ala Thr Leu Ala Trp Met Val Ile Met Gly Asp Gly Leu His 590 595600 Asn Phe Ser Asp Gly Leu Ala Ile Gly Ala Ala Phe Thr Glu Gly 605 610615 Leu Ser Ser Gly Leu Ser Thr Ser Val Ala Val Phe Cys His Glu 620 625630 Leu Pro His Glu Leu Gly Asp Phe Ala Val Leu Leu Lys Ala Gly 635 640645 Met Thr Val Lys Gln Ala Val Leu Tyr Asn Ala Leu Ser Ala Met 650 655660 Leu Ala Tyr Leu Gly Met Ala Thr Gly Ile Phe Ile Gly His Tyr 665 670675 Ala Glu Asn Val Ser Met Trp Ile Phe Ala Leu Thr Ala Gly Leu 680 685690 Phe Met Tyr Val Ala Leu Val Asp Met Val Pro Glu Met Leu His 695 700705 Asn Asp Ala Ser Asp His Gly Cys Ser Arg Trp Gly Tyr Phe Phe 710 715720 Leu Gln Asn Ala Gly Met Leu Leu Gly Phe Gly Ile Met Leu Leu 725 730735 Ile Pro Tyr Leu Asn Ile Lys Ser Cys Ser Tyr Lys Phe Leu Val 740 745750 Lys Val 3 2776 DNA Homo sapien 3 ccggccgtgt ggaaccaaac ctgcgcgcgtggccgggccg tgggacaacg 50 aggccgcgga gacgaaggcg caatggcgag gaagttatctgtaatcttga 100 tcctgacctt tgccctctct gtcacaaatc cccttcatga actaaaagca150 gctgctttcc cccagaccac tgagaaaatt agtccgaatt gggaatctgg 200cattaatgtt gacttggcaa tttccacacg gcaatatcat ctacaacagc 250 ttttctaccgctatggagaa aataattctt tgtcagttga agggttcaga 300 aaattacttc aaaatataggcatagataag attaaaagaa tccatataca 350 ccatgaccac gaccatcact cagaccacgagcatcactca gaccatgagc 400 gtcactcaga ccatgagcat cactcagacc acgagcatcactctgaccat 450 gatcatcact cccaccataa tcatgctgct tctggtaaaa ataagcgaaa500 agctctttgc ccagaccatg actcagatag ttcaggtaaa gatcctagaa 550acagccaggg gaaaggagct caccgaccag aacatgccag tggtagaagg 600 aatgtcaaggacagtgttag tgctagtgaa gtgacctcaa ctgtgtacaa 650 cactgtctct gaaggaactcactttctaga gacaatagag actccaagac 700 ctggaaaact cttccccaaa gatgtaagcagctccactcc acccagtgtc 750 acatcaaaga gccgggtgag ccggctggct ggtaggaaaacaaatgaatc 800 tgtgagtgag ccccgaaaag gctttatgta ttccagaaac acaaatgaaa850 atcctcagga gtgtttcaat gcatcaaagc tactgacatc tcatggcatg 900ggcatccagg ttccgctgaa tgcaacagag ttcaactatc tctgtccagc 950 catcatcaaccaaattgatg ctagatcttg tctgattcat acaagtgaaa 1000 agaaggctga aatccctccaaagacctatt cattacaaat agcctgggtt 1050 ggtggtttta tagccatttc catcatcagtttcctgtctc tgctgggggt 1100 tatcttagtg cctctcatga atcgggtgtt tttcaaatttctcctgagtt 1150 tccttgtggc actggccgtt gggactttga gtggtgatgc ttttttacac1200 cttcttccac attctcatgc aagtcaccac catagtcata gccatgaaga 1250accagcaatg gaaatgaaaa gaggaccact tttcagtcat ctgtcttctc 1300 aaaacatagaagaaagtgcc tattttgatt ccacgtggaa gggtctaaca 1350 gctctaggag gcctgtatttcatgtttctt gttgaacatg tcctcacatt 1400 gatcaaacaa tttaaagata agaagaaaaagaatcagaag aaacctgaaa 1450 atgatgatga tgtggagatt aagaagcagt tgtccaagtatgaatctcaa 1500 ctttcaacaa atgaggagaa agtagataca gatgatcgaa ctgaaggcta1550 tttacgagca gactcacaag agccctccca ctttgattct cagcagcctg 1600cagtcttgga agaagaagag gtcatgatag ctcatgctca tccacaggaa 1650 gtctacaatgaatatgtacc cagagggtgc aagaataaat gccattcaca 1700 tttccacgat acactcggccagtcagacga tctcattcac caccatcatg 1750 actaccatca tattctccat catcaccaccaccaaaacca ccatcctcac 1800 agtcacagcc agcgctactc tcgggaggag ctgaaagatgccggcgtcgc 1850 cactttggcc tggatggtga taatgggtga tggcctgcac aatttcagcg1900 atggcctagc aattggtgct gcttttactg aaggcttatc aagtggttta 1950agtacttctg ttgctgtgtt ctgtcatgag ttgcctcatg aattaggtga 2000 ctttgctgttctactaaagg ctgacatgac cgttaagcag gctgtccttt 2050 ataatgcatt gtcagccatgctggcgtatc ttggaatggc aacaggaatt 2100 ttcattggtc attatgctga aaatgtttctatgtggatat ttgcacttac 2150 tgctggctta ttcatgtatg ttgctctggt tgatatggtacctgaaatgc 2200 tgcacaatga tgctagtgac catggatgta gccgctgggg gtatttcttt2250 ttacagaatg ctgggatgct tttgggtttt ggaattatgt tacttatttc 2300catatttgaa cataaaatcg tgtttcgtat aaatttctag ttaaggttta 2350 aatgctagagtagcttaaaa agttgtcata gtttcagtag gtcataggga 2400 gatgagtttg tatgctgtactatgcagcgt ttaaagttag tgggttttgt 2450 gatttttgta ttgaatattg ctgtctgttacaaagtcagt taaaggtacg 2500 ttttaatatt taagttattc tatcttggag ataaaatctgtatgtgcaat 2550 tcaccggtat taccagttta ttatgtaaac aagagatttg gcatgacatg2600 ttctgtatgt ttcagggaaa aatgtcttta atgctttttc aagaactaac 2650acagttattc ctatactgga ttttaggtct ctgaagaact gctggtgttt 2700 aggaataagaatgtgcatga agcctaaaat accaagaaag cttatactga 2750 atttaagcaa aaaaaaaaaaaaaaaa 2776 4 755 PRT Homo sapien 4 Met Ala Arg Lys Leu Ser Val Ile LeuIle Leu Thr Phe Ala Leu 1 5 10 15 Ser Val Thr Asn Pro Leu His Glu LeuLys Ala Ala Ala Phe Pro 20 25 30 Gln Thr Thr Glu Lys Ile Ser Pro Asn TrpGlu Ser Gly Ile Asn 35 40 45 Val Asp Leu Ala Ile Ser Thr Arg Gln Tyr HisLeu Gln Gln Leu 50 55 60 Phe Tyr Arg Tyr Gly Glu Asn Asn Ser Leu Ser ValGlu Gly Phe 65 70 75 Arg Lys Leu Leu Gln Asn Ile Gly Ile Asp Lys Ile LysArg Ile 80 85 90 His Ile His His Asp His Asp His His Ser Asp His Glu HisHis 95 100 105 Ser Asp His Glu Arg His Ser Asp His Glu His His Ser AspHis 110 115 120 Glu His His Ser Asp His Asp His His Ser His His Asn HisAla 125 130 135 Ala Ser Gly Lys Asn Lys Arg Lys Ala Leu Cys Pro Asp HisAsp 140 145 150 Ser Asp Ser Ser Gly Lys Asp Pro Arg Asn Ser Gln Gly LysGly 155 160 165 Ala His Arg Pro Glu His Ala Ser Gly Arg Arg Asn Val LysAsp 170 175 180 Ser Val Ser Ala Ser Glu Val Thr Ser Thr Val Tyr Asn ThrVal 185 190 195 Ser Glu Gly Thr His Phe Leu Glu Thr Ile Glu Thr Pro ArgPro 200 205 210 Gly Lys Leu Phe Pro Lys Asp Val Ser Ser Ser Thr Pro ProSer 215 220 225 Val Thr Ser Lys Ser Arg Val Ser Arg Leu Ala Gly Arg LysThr 230 235 240 Asn Glu Ser Val Ser Glu Pro Arg Lys Gly Phe Met Tyr SerArg 245 250 255 Asn Thr Asn Glu Asn Pro Gln Glu Cys Phe Asn Ala Ser LysLeu 260 265 270 Leu Thr Ser His Gly Met Gly Ile Gln Val Pro Leu Asn AlaThr 275 280 285 Glu Phe Asn Tyr Leu Cys Pro Ala Ile Ile Asn Gln Ile AspAla 290 295 300 Arg Ser Cys Leu Ile His Thr Ser Glu Lys Lys Ala Glu IlePro 305 310 315 Pro Lys Thr Tyr Ser Leu Gln Ile Ala Trp Val Gly Gly PheIle 320 325 330 Ala Ile Ser Ile Ile Ser Phe Leu Ser Leu Leu Gly Val IleLeu 335 340 345 Val Pro Leu Met Asn Arg Val Phe Phe Lys Phe Leu Leu SerPhe 350 355 360 Leu Val Ala Leu Ala Val Gly Thr Leu Ser Gly Asp Ala PheLeu 365 370 375 His Leu Leu Pro His Ser His Ala Ser His His His Ser HisSer 380 385 390 His Glu Glu Pro Ala Met Glu Met Lys Arg Gly Pro Leu PheSer 395 400 405 His Leu Ser Ser Gln Asn Ile Glu Glu Ser Ala Tyr Phe AspSer 410 415 420 Thr Trp Lys Gly Leu Thr Ala Leu Gly Gly Leu Tyr Phe MetPhe 425 430 435 Leu Val Glu His Val Leu Thr Leu Ile Lys Gln Phe Lys AspLys 440 445 450 Lys Lys Lys Asn Gln Lys Lys Pro Glu Asn Asp Asp Asp ValGlu 455 460 465 Ile Lys Lys Gln Leu Ser Lys Tyr Glu Ser Gln Leu Ser ThrAsn 470 475 480 Glu Glu Lys Val Asp Thr Asp Asp Arg Thr Glu Gly Tyr LeuArg 485 490 495 Ala Asp Ser Gln Glu Pro Ser His Phe Asp Ser Gln Gln ProAla 500 505 510 Val Leu Glu Glu Glu Glu Val Met Ile Ala His Ala His ProGln 515 520 525 Glu Val Tyr Asn Glu Tyr Val Pro Arg Gly Cys Lys Asn LysCys 530 535 540 His Ser His Phe His Asp Thr Leu Gly Gln Ser Asp Asp LeuIle 545 550 555 His His His His Asp Tyr His His Ile Leu His His His HisHis 560 565 570 Gln Asn His His Pro His Ser His Ser Gln Arg Tyr Ser ArgGlu 575 580 585 Glu Leu Lys Asp Ala Gly Val Ala Thr Leu Ala Trp Met ValIle 590 595 600 Met Gly Asp Gly Leu His Asn Phe Ser Asp Gly Leu Ala IleGly 605 610 615 Ala Ala Phe Thr Glu Gly Leu Ser Ser Gly Leu Ser Thr SerVal 620 625 630 Ala Val Phe Cys His Glu Leu Pro His Glu Leu Gly Asp PheAla 635 640 645 Val Leu Leu Lys Ala Asp Met Thr Val Lys Gln Ala Val LeuTyr 650 655 660 Asn Ala Leu Ser Ala Met Leu Ala Tyr Leu Gly Met Ala ThrGly 665 670 675 Ile Phe Ile Gly His Tyr Ala Glu Asn Val Ser Met Trp IlePhe 680 685 690 Ala Leu Thr Ala Gly Leu Phe Met Tyr Val Ala Leu Val AspMet 695 700 705 Val Pro Glu Met Leu His Asn Asp Ala Ser Asp His Gly CysSer 710 715 720 Arg Trp Gly Tyr Phe Phe Leu Gln Asn Ala Gly Met Leu LeuGly 725 730 735 Phe Gly Ile Met Leu Leu Ile Ser Ile Phe Glu His Lys IleVal 740 745 750 Phe Arg Ile Asn Phe 755 5 391 DNA Artificial sequenceProbe 5 tttttttttg atataaggaa tttattgtgt aaaaaagaaa catctagaga 50atgccacaga caggcctagt atggctacag taccgtatat aaaagacaat 100 tgctcacaatgatagcactg aagcactgag agatatcaaa gtactttctg 150 aatcgaatca aatgatacttagtgtagttt taatatcctc atatatatca 200 aagttttact actctgataa ttttgtaaaccagggtaacc aggancatcc 250 agtcatacag cttttgggtg atatataact tgggcaataacccagtctgg 300 gtgatacnta aanctactca ctgtactcat ctgggtatat acccgcacgg350 ancattttgg aaattcccaa tttcttgggt caggtgatat a 391 6 28 DNAArtificial sequence Primer 6 atgttgactt ggcaatttcc acacggca 28 7 27 DNAArtificial sequence Primer 7 taatgccaga ttcccaattc ggactaa 27 8 46 DNAArtificial sequence Probe 8 ttagttcatg aaggggattt gtgacagaga gggcaaaggtcaggat 46 9 42 DNA Artificial sequence Primer 9 caacatcaaa tgcatcaacttcatgaacta aaagcagctg ct 42 10 43 DNA Artificial sequence Primer 10gagctcgagc ggccgcttag gtctttggag ggatttcagc ctt 43 11 17 PRT Artificialsequence Leader sequence 11 Met Lys His Gln His Gln His Gln His Gln HisGln His Gln Met 1 5 10 15 His Gln 12 551 DNA Artificial sequence CDNA 12tgccattcac atttccacga tacactcggc cagtcagacg atctcattca 50 ccaccatcatgactaccatc atattctcca tcatcaccac caccaaaacc 100 accatcctca cagtcacagccagcgctact ctcgggagga gctgaaagat 150 gccggcgtcg ccactttggc ctggatggtgataatgggtg atggcctgca 200 caatttcagc gatggcctag caattggtgc tgcttttactgaaggcttat 250 caagtggttt aagtacttct gttgctgtgt tctgtcatga gttgcctcat300 gaattaggtg actttgctgt tctactaaag gctgacatga ccgttaagca 350ggctgtcctt tataatgcat tgtcagccat gctggcgtat cttggaatgg 400 caacaggaattttcattggt cattatgctg aaaatgtttc tatgtggata 450 tttgcactta ctgctggcttattcatgtat gttgctctgg ttgatatggt 500 acctgaaatg ctgcacaatg atgctagtgaccatggatgt agccgctggg 550 g 551 13 48 DNA Artificial sequence Primer 13ggattctaat acgactcact atagggctgc cattcacatt tccacgat 48 14 44 DNAArtificial sequence Primer 14 ctatgaaatt aaccctcact aaagggaccccagcgcctac atcc 44 15 620 DNA Artificial sequence CDNA 15 tggtcgtggtcttgggggtg gtctttggga tcctcatcaa gcgacggcag 50 cagaagatcc ggaagtacacgatgcggaga ctgctgcagg aaacggagct 100 ggtggagccg ctgacaccta gcggagcgatgcccaaccag gcgcagatgc 150 ggatcctgaa agagacggag ctgaggaagg tgaaggtgcttggatctggc 200 gcttttggca cagtctacaa gggcatctgg atccctgatg gggagaatgt250 gaaaattcca gtggccatca aagtgttgag ggaaaacaca tcccccaaag 300ccaacaaaga aatcttagac gaagcatacg tgatggctgg tgtgggctcc 350 ccatatgtctcccgccttct gggcatctgc ctgacatcca cggtgcagct 400 ggtgacacag cttatgccctatggctgcct cttagaccat gtccgggaaa 450 accgcggacg cctgggctcc caggacctgctgaactggtg tatgcagatt 500 gccaagggga tgagctacct ggaggatgtg cggctcgtacacagggactt 550 ggccgctcgg aacgtgctgg tcaagagtcc caaccatgtc aaaattacag600 acttcgggct ggctcggctg 620 16 290 DNA Artificial sequence CDNA 16gctgcctgac ggccaggtca tcaccattgg caatgagcgg ttccgctgcc 50 ctgaggcactcttccagcct tccttcctgg gcatggagtc ctgtggcatc 100 cacgaaacta ccttcaactccatcatgaag tgtgactgtg acatccgcaa 150 agacctgtac gccaacacag tgctgtctggcggcaccacc atgtaccctg 200 gcattgccga caggatgcag aaggagatca ctgccctggcacccagcaca 250 atgaagatca agatcattgc tcctctgagc gcaagtactc 290 17 7 PRTHomo sapien 17 His Asp His His Ser His His 1 5 18 13 PRT Homo sapien 18Ser Ile Phe Glu His Lys Ile Val Phe Arg Ile Asn Phe 1 5 10 19 22 PRTHomo sapien 19 His Glu His His Ser Asp His Glu His His Ser Asp His AspHis 1 5 10 15 His Ser His His Asn His Ala 20

What is claimed is:
 1. An isolated nucleic acid comprising a sequencehaving at least 65% homology to a sequence from nucleotide 412 to andincluding nucleotide 477 of SEQ ID NO:3 or its complementary sequence.2. The isolated nucleic acid of claim 1, wherein the homology is atleast 75%.
 3. The isolated nucleic acid of claim 1, wherein the homologyis at least 85%.
 4. The isolated nucleic acid of claim 1, wherein thehomology is at least 90%.
 5. The isolated nucleic acid of claim 1,wherein the homology is at least 96%.
 6. The isolated nucleic acid ofclaim 1 comprising the sequence from nucleotide 412 to and includingnucleotide 477 of SEQ ID NO:3 or its complementary sequence.
 7. Theisolated nucleic acid of claim 1 comprising SEQ ID NO:3.
 8. The isolatednucleic acid of claim 1 comprising a sequence from nucleotide 446 to andincluding nucleotide 463 of SEQ ID NO:3 or its complementary sequence.9. The isolated nucleic acid of claim 1, wherein the nucleic acidencodes an antigenic polypeptide.
 10. The isolated nucleic acid of claim1, wherein the sequence encodes an extracellular domain.
 11. An isolatedpolypeptide comprising a sequence having at least 65% homology to SEQ IDNO:19.
 12. The isolated polypeptide of claim 11, wherein the homology isat least 75%.
 13. The isolated polypeptide of claim 11, wherein thehomology is at least 85%.
 14. The isolated polypeptide of claim 11,wherein the homology is at least 90%.
 15. The isolated polypeptide ofclaim 11, wherein the homology is at least 96%.
 16. The isolatedpolypeptide of claim 11 comprising SEQ ID NO:19.
 17. The isolatedpolypeptide of claim 11 comprising a sequence having at least 50%homology to SEQ ID NO:17.
 18. The isolated polypeptide of claim 11comprising SEQ ID NO:17.
 19. An isolated polypeptide comprising asequence having at least 20% homology to SEQ ID NO:18.
 20. The isolatedpolypeptide of claim 11 comprising SEQ ID NO:4.
 21. The isolatedpolypeptide of claim 11, wherein the polypeptide is antigenic.
 22. Theisolated polypeptide of claim 11, wherein the sequence comprises aportion of an extracellular domain.
 23. The isolated polypeptide ofclaim 11, wherein the polypeptide is aqueous soluble.
 24. An isolatedpolypeptide having at least 98% homology to the sequence from amino acid1 to and including amino acid 327 of SEQ ID NO:4.
 25. An isolatedpolypeptide, wherein the polypeptide binds to a monoclonal antibody,wherein the antibody binds to the same epitope to which the antibodyproduced by hybridoma cell line deposited under American Type CultureCollection Accession Number ATCC ______ (LIV-1.2945.2G1.1C7.2F10) binds.26. An isolated polypeptide, wherein the polypeptide binds to amonoclonal antibody, wherein the antibody binds to the same epitope towhich the antibody produced by hybridoma cell line deposited underAmerican Type Culture Collection Accession Number ATCC ______(LIV-1.2982.4A12.1E8.1C4) binds.
 27. An isolated polypeptide, whereinthe polypeptide binds to a monoclonal antibody, wherein the antibodybinds to the same epitope to which the antibody produced by hybridomacell line deposited under American Type Culture Collection AccessionNumber ATCC ______ (LIV-1.2983.3G9.1D4.1D7) binds.
 28. An isolatedpolypeptide, wherein the polypeptide binds to a monoclonal antibody,wherein the antibody binds to the same epitope to which the antibodyproduced by hybridoma cell line deposited under American Type CultureCollection Accession Number ATCC ______ (LIV-1.2984.6D6.1H10.2C1) binds.29. An isolated polypeptide, wherein the polypeptide binds to amonoclonal antibody, wherein the antibody binds to the same epitope towhich the antibody produced by hybridoma cell line deposited underAmerican Type Culture Collection Accession Number ATCC ______(LIV-1.2985.4F3.2D6.1D7) binds.
 30. An isolated polypeptide, wherein thepolypeptide binds to a monoclonal antibody, wherein the antibody bindsto the same epitope to which the antibody produced by hybridoma cellline deposited under American Type Culture Collection Accession NumberATCC ______ (LIV-1.2987.1D8.1C11.2B7) binds.
 31. An isolatedpolypeptide, wherein the polypeptide binds to a monoclonal antibody,wherein the antibody binds to the same epitope to which the antibodyproduced by hybridoma cell line deposited under American Type CultureCollection Accession Number ATCC ______ (LIV-1.2988.1A7.1F2.1H7) binds.32. A monoclonal antibody that binds a polypeptide, wherein thepolypeptide comprises a sequence that is at least 80% homologous to thesequence from amino acid 1 to and including amino acid 327 of SEQ IDNO:4.
 33. The monoclonal antibody of claim 32, wherein the homology isat least 98%.
 34. A monoclonal antibody that binds a polypeptide,wherein the polypeptide comprises a sequence that is at least 65%homologous to SEQ ID NO:19.
 35. The monoclonal antibody of claim 32,wherein the homology is at least 75%.
 36. The monoclonal antibody ofclaim 32, wherein the homology is at least 85%.
 37. The monoclonalantibody of claim 32, wherein the homology is at least 90%.
 38. Themonoclonal antibody of claim 32, wherein the homology is at least 96%.39. The monoclonal antibody of claim 32, wherein the polypeptidecomprises SEQ ID NO:19.
 40. The monoclonal antibody of claim 32, whereinthe polypeptide comprises SEQ ID NO:17.
 41. The monoclonal antibody ofclaim 32, wherein the antibody is a LIV-1-164647 antagonist.
 42. Themonoclonal antibody of claim 32, wherein the antibody inhibitsproliferation of tumor cells.
 43. The monoclonal antibody of claim 42,wherein the tumors cells are selected from the group consisting ofbreast, lung, prostate, colon, ovary, uterus, kidney, gastric, andsalivary gland carcinomas, or other tumor cell types expressing theLIV-1-164647 protein.
 44. The monoclonal antibody as in claim 42,wherein the antibody inhibits in vivo proliferation of tumor cells thatoverexpress LIV-1.
 45. The monoclonal antibody as in claim 32, whichantibody is a murine monoclonal antibody.
 46. The monoclonal antibody asin claim 32, which antibody is a murine-human hybrid antibody.
 47. Themonoclonal antibody as in claim 32, wherein the antibody competes forbinding to the ligand binding site of a ligand of the LIV-1 protein. 48.The monoclonal antibody as in claim 32, wherein the antibody reducesexpression LIV-1.
 49. The monoclonal antibody as in claim 32, whereinthe antibody activates complement.
 50. The monoclonal antibody as inclaim 32, wherein the antibody mediates antibody dependent cellularcytotoxicity.
 51. A monoclonal antibody that binds to an epitope,wherein the epitope comprises an amino acid sequence having at least 65%homology to SEQ ID NO:19.
 52. The monoclonal antibody of claim 49,wherein the epitope comprises an amino acid sequence having at least 80%homology to SEQ ID NO:17.
 53. The monoclonal antibody of claim 52,wherein the epitope comprises SEQ ID NO:17.
 54. A monoclonal antibody,wherein the antibody binds to the same epitope as the epitope to whichthe monoclonal antibody produced by a hybridoma cell line binds, whereinthe hybridoma cell line is selected from the group consisting ofhybridoma cell lines deposited under American Type Culture CollectionAccession Numbers ATCC ______ (LIV-1.2945.2G1.1C7.2F10); ATCC ______(LIV-1.2982.4A12.1E8.1C4); ATCC ______ (LIV-1.12983.3C9.1D4.1D7); ATCC______ (LIV-1.2984.6D6.1H10.2C1); ATCC ______ (LIV-1.2985.4F3.2D6.1D7);ATCC ______ (LIV-1.2987.1D8.1C11.2B7); ATCC ______(LIV-1.2988.1A7.1F2.1H7).
 55. An immunotoxin which is a conjugate of acytotoxic moiety and the monoclonal antibody of claim
 32. 56. Ahybridoma producing the monoclonal antibody of claim
 32. 57. A hybridomaselected from the group consisting of ATCC ______(LIV-1.2945.2G1.1C7.2F10), ATCC ______ (LIV-1.2982-4A12.1E8.1C4); ATCC______ (LIV-1.2983.3G9.1D4.1D7); ATCC ______ (LIV-1.2984.6D6.1H10.2C1);ATCC ______ (LIV-1.2985.4F3.2D6.1D7); ATCC ______(LIV-1.2987.1D8.1C10.2B7); ATCC ______ (LIV-1.2988.1A7.1F2.1H7).
 58. Anassay for detecting a tumor, the method comprising: (a) exposing asample from a mammal to an antibody that specifically binds apolypeptide comprising a sequence having at least 80% homologous to asequence from amino acid 1 to and including amino acid 327 of SEQ IDNO:4; and (b) determining the extent of binding to a polypeptide in thesample.
 59. The assay of claim 58, wherein the polypeptide in the sampleis on a cell from the mammal.
 60. The assay of claim 58, wherein thesample is body fluid from the mammal and the polypeptide is a solublepolypeptide.
 61. The assay as in claim 58, wherein the antibody is amonoclonal antibody.
 62. The assay as in claim 58, wherein the assay isperformed in vitro and the assay in an ELISA assay.
 63. The assay as inclaim 59, wherein the cell is a plurality of cells which remain withinthe body of a mammal, the antibody is a plurality of antibodies whichare tagged with a radioactive isotope and administered to the mammal,and the extent of binding of the antibodies to the cells is observed byexternal scanning for radioactivity.
 64. The assay as in claim 58,wherein the assay further comprises anti-ErbB2 antibodies tagged with asecond radioactive isotope and administered to the mammal, and theextent of bindings of the antibodies to the cells by the taggedanti-ErbB2 antibodies to the cells is observed by external scanning forradioactivity.
 65. A method of inhibiting the growth of tumor cells, themethod comprising: administering to a mammal a therapeutically effectiveamount of an antibody capable of binding to an amino acid sequencehaving at least 80% homology to a sequence from amino acid 1 to andincluding amino acid 327 of SEQ ID NO:4, which antibody inhibitsproliferation of LIV-1-164647-expressing cells.
 66. The method of claim65, wherein the antibodies activate complement.
 67. The method of claim65, wherein the antibodies mediate antibody dependent cellularcytotoxicity.
 68. The method of claim 65, wherein the antibodies aremonoclonal antibodies.
 69. The method of claim 65, wherein the tumorcells comprise a carcinoma selected from breast, lung, prostate, colon,ovary, uterus, kidney, gastric, and salivary eland carcinomas, or othertumor cell types expressing the LIV-1-164647 protein.
 70. The method ofclaim 69, wherein the tumor cells are breast tissue cells.
 71. Themethod of claim 69, wherein the mammal is a human.
 72. The method ofclaim 65, wherein the method further comprises administering atherapeutically effective amount of a cytotoxic factor.
 73. The methodof claim 72, wherein the antibodies and cytotoxic factor areadministered simultaneously.
 74. The method of claim 72, wherein theantibody is administered to the patient before the cytoxic agent. 75.The method of claim 72, wherein the cytotoxic agent is administeredbefore the antibody.
 76. The method of claim 72, wherein the cytotoxicagent is conjugated to the antibody.
 77. A composition comprising anantibody that binds specifically to an amino acid sequence having atleast 80% homologous to a sequence from amino acid 1 to and includingamino acid 327 of SEQ ID NO:4, and a pharmaceutically acceptablecarrier.
 78. A composition comprising an antibody and a pharmaceuticallyacceptable carrier, wherein the antibody is a monoclonal antibodyproduced by a hybridoma cell line selected from the group consisting ofATCC ______ (LIV-1.2945.2G1.1C7.2F10); ATCC ______(LIV-1.2982.4A12.1E8.1C4); ATCC ______ (LIV-12983.3G9.1D4.1D7); ATCC______ (LIV-1.2984.6D6.1H10.2C1); ATCC ______ (LIV-1.2985.4F3.2D6.1D7);ATCC ______ (LIV-1.2987.1D8.1C10.2B7); ATCC ______(LIV-1.2988.1A7.1F2.1H7).
 79. The composition of claim 77, furthercomprising a cytotoxic factor.
 80. A method of diagnosing tumor in amammal, comprising detecting the level of expression of a nucleic acidsequence comprising a sequence having at least 65% homology to asequence from nucleotide 412 to and including nucleotide 477 of SEQ IDNO:3 or its complementary sequence in a test sample obtained from themammal, and in a control sample, wherein a higher expression level inthe test sample indicates the presence of tumor in the mammal from whichthe test sample was obtained.
 81. A method of diagnosing tumor in amammal, comprising: (a) contacting an antibody with a test sampleobtained from the mammal; and (b) detecting the formation of a complexbetween the antibody and a polypeptide of the test sample, wherein theantibody binds the polypeptide, the polypeptide comprising an amino acidsequence having at least 80% homology to the sequence from amino acid 1to and including amino acid 327 of SEQ ID NO:4.
 82. The method of claim80, wherein said test sample is obtained from an individual suspected tohave neoplastic cell growth or proliferation.
 83. The method of claim81, wherein the test sample is obtained from an individual suspected tohave neoplastic cell growth or proliferation.
 84. An article ofmanufacture comprising: a container; and a composition of claim 77contained within the container.
 85. An article of manufacturecomprising: a container; and a composition of claim 78 contained withthe container.
 86. An article of manufacture, comprising: a container; alabel on the container; and the composition of claim 77, wherein thecomposition is effective for inhibiting the growth of tumor cells, andthe label on the container indicates that the composition can be usedfor treating conditions characterized by overexpression of aLIV-1-164647 polypeptide.
 87. An article of manufacture, comprising: acontainer; a label on the container; and the composition of claim 78,wherein the composition is effective for inhibiting the growth of tumorcells, and the label on the container indicates that the composition canbe used for treating conditions characterized by overexpression of aLIV-1-164647 polypeptide.
 88. A method for identifying a compoundcapable of inhibiting the expression or activity of a LIV-1-164647polypeptide, comprising contacting a candidate compound with apolypeptide comprising a sequence having at least 65% homology to SEQ IDNO:19.
 89. The method of claim 88 wherein the candidate compound or thepolypeptide is immobilized on a solid support.
 90. The method of claim89 wherein the non-immobilized component carries a detectable label. 91.A host cell comprising a nucleic acid sequence having at least 65%nucleic acid sequence homology to the sequence from nucleotide 412 toand including nucleotide 477 of SEQ ID NO:3 or its complementarysequence.
 92. The host cell of claim 91, wherein the cell is a Chinesehamster ovary (CHO) cell.
 93. The host cell of claim 91, wherein thecell is an E. coli.
 94. The host cell of claim 91, wherein the cell is ayeast cell.
 95. The host cell of claim 91, wherein the cell is an insectcell.
 96. The antibody of claim 32, wherein the antibody is a chimericantibody.
 97. The antibody of claim 32, wherein the antibody is ahumanized antibody.
 98. The antibody of claim 32, wherein the antibodyis a human antibody.
 99. The antibody of claim 32, wherein the antibodyis an antibody fragment selected from the gropu consisting of Fab, Fab′,F(ab′)₂, Fv fragments, diabodies, single chain antibodies, andmultispecific antibodies.
 100. A host cell which produces the antibodyof claim 32, wherein the host cell is selected from the group consistingof a Chinese Hamster Ovary (CHO) cell, E. coli, yeast cell, and insectcell.